Isolated nuclease and use thereof

Abstract

It relates to the field of molecular biology, and specifically to an isolated nuclease and the use thereof. It further specifically relates to: a nucleic acid and a nucleic acid construct encoding the nuclease, a guide RNA and a nucleic acid construct thereof, and a composition, a recombinant vector, a recombinant host cell and a kit comprising the nuclease. It further specifically relates to: a method for introducing a double-strand break into a targeting gene of a host cell, a method for deleting, replacing or inserting a targeting gene of a host cell, and a method for obtaining a host cell in which a targeting gene is deleted, replaced or inserted.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims priority to Chinese Patent Application No. 202310304837.4 filed on Mar. 27, 2023, and PCT Application No. PCT / CN2023 / 135175 filed on Nov. 29, 2023, the entire contents of which are hereby incorporated by reference in their entirety for all purpose.TECHNICAL FIELD

[0002] The present application relates to the field of molecular biology, and specifically to an isolated nuclease and the use thereof. The present application further specifically relates to: a nucleic acid and a nucleic acid construct encoding the nuclease, a guide RNA and a nucleic acid construct thereof, and a composition, a recombinant vector, a recombinant host cell and a kit comprising the nuclease. The present application further specifically relates to: a method for introducing a double-strand break into a targeting gene of a host cell, a method for deleting, replacing or inserting a targeting gene of a host cell, and a method for obtaining a host cell in which a targeting gene is deleted, replaced or inserted. The present application further specifically relates to the use of the nuclease, the nucleic acid and the nucleic acid construct encoding the nuclease, the guide RNA and the nucleic acid construct thereof, the composition, the recombinant vector, or the recombinant host cell for introducing a double-strand break into a targeting gene of a host cell, deleting, replacing or inserting a targeting gene of a host cell, and preparing a drug or a preparation for gene therapy, cell therapy, genome research, and stem cell induction and post-induction differentiation.BACKGROUND

[0003] With the rapid development of modern biotechnology and the advent of post-genome era, people are entering the stage of rewriting or even redesigning genetic information from the stage of reading biological genetic DNA information. The discovery of CRISPR / Cas9 technology has made a revolutionary breakthrough in gene editing technology. CRISPR / Cas9 is an RNA-mediated targeted gene editing tool, which can specifically recognize and cleave different endogenous DNA sequences through reprogramming of sgRNA. Cas9 has two nuclease domains, RuvC and HNH, which are responsible for the cleavage of either strand of DNA respectively. Mutating either of these sites can convert Cas9 into a single-strand Cas9 nickase. Important new technologies concerning Cas9, such as base editing and prime editing, are all designed based on Cas9 nickase.

[0004] However, some shortcomings of CRISPR / Cas9 limit its application: First, the CDS sequence of spCas9 has a length exceeding 4.1 Kb, which exceeds the maximum effective packaging capacity of adenovirus (AAV), and therefore it is difficult for the adenovirus-mediated gene delivery; although lentivirus has a stronger packaging capacity than AAV (with an upper loading limit of about 9 kb), the proportion of proteins in spCas9 is still too high, limiting the potential for subsequent engineering. These shortcomings seriously restrict the application of spCas9 in clinical medicine. Subsequently, CRISPR / Cas12 or 12f system with a smaller molecular weight appears, but the editing efficiency of proteins such as Cas12 is not superior to that of spCas9. Therefore, spCas9 is still widely accepted and used at present. Second, the PAM sequence of spCas9, which is the NGG sequence, is relatively simple and has a higher occurrence rate in the genome. Its advantage lies in the flexibility in reprograming sgRNA to complete the recognition and cleavage of different DNA sequences. However, this flexibility also leads to the off-target effects of suboptimal genome editing outcomes.

[0005] Therefore, gene editing technologies realized using RNA-mediated endonuclease, i.e., insertion sequences IscB and TnpB from IS200 / IS605 family, appear subsequently. They are widely distributed in microorganisms and have a more compact protein structure, with a size of about 400 aa that is less than ⅓ of spCas9, so they have greater potential for engineering in terms of the application of enzymes. TnpB cleaves DNA next to the 5′ TTGAT transposon-associated motif (TAM) through reRNA (right element RNA, derived from RE element in ISDra2 transposon) mediation, thereby breaking and mutating the DNA sequence in the genome. The DNA cleavage function of TnpB needs to meet two conditions at the same time: (1) TAM sequence; (2) a sequence located at the 3′ end of reRNA that matches with a targeting gene. Different nucleases can recognize different TAM, and therefore the excavation of more highly active nuclease tools and the verification and detection of their functions can provide more, better and flexible choices for the development of gene editing strategies.

[0006] It should be noted that methods described in this section are not necessarily methods that have been previously conceived or employed. It should not be assumed that any of the methods described in this section is considered to be the prior art just because they are included in this section, unless otherwise indicated expressly. Similarly, the problem mentioned in this section should not be considered to be universally recognized in any prior art, unless otherwise indicated expressly.SUMMARY

[0007] In order to solve the above problems, the present application is intended to find RNA-mediated endonucleases having a suitable protein molecular weight and good gene editing effects, and provide more diverse and specific tools for gene editing.

[0008] The present application provides an isolated nuclease, wherein the nuclease comprises an amino acid sequence as shown in the following formula:(X1)(X2)a(X3)(X4)(X5)b(X6)(X7)c(X8)(X9)d(X10)(X11)e(X12)(X13)f(X14)(X15)g(X16)wherein a, b, c, d, e, f, and g are the numbers of amino acids; (X1), (X3), (X4), (X6), (X8), (X10), (X12), (X14), and (X16) are independently polar amino acids or aliphatic amino acids; (X2) is any amino acid, and a is 15 or 16; (X5) is any amino acid, and b is 2; (X7) is any amino acid, and c is 2, 3 or 4; (X9) is any amino acid, and d is 14, 15, 16, 17 or 18; (X11) is any amino acid, and e is 1 or 2; (X13) is any amino acid, and f is 6; and (X15) is any amino acid, and g is 5.

[0010] According to an embodiment of the present application, an isolated nuclease can be provided, wherein the nuclease has a nuclease sequence selected from the following (i) or a variant sequence of the aforementioned nuclease having a nuclease activity in (ii)-(iv): (i) at least one amino acid sequence as shown in any one of SEQ ID NOs: 1-197; (ii) at least one of sequences obtained by performing deletion, substitution, insertion, or mutation of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids on the amino acid sequence as shown in any one of SEQ ID NOs: 1-197; (iii) at least one of amino acid sequences having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence as shown in any one of SEQ ID NOs: 1-197; and (iv) at least one of sequences obtained by further fusing the amino acid sequence as shown in any one of SEQ ID NOs: 1-197 with other sequences.

[0011] According to an embodiment of the present application, a guide RNA can be provided, wherein the guide RNA comprises a reRNA, the reRNA comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 198-394 or a variant thereof, and the guide RNA can bind to a specific nuclease.

[0012] According to an embodiment of the present application, a nucleic acid can be provided, wherein, the nucleic acid encodes the nuclease described in the present application and / or the guide RNA described in the present application.

[0013] According to an embodiment of the present application, a nucleic acid construct can be provided, comprising the nucleic acid described in the present application, and further comprising a promoter.

[0014] According to an embodiment of the present application, a composition may be provided, wherein, the composition includes: an IS200 / IS605 family nuclease or a functional fragment thereof, or comprises a nucleic acid encoding the IS200 / IS605 family nuclease or the functional fragment thereof, and the nuclease or the functional fragment thereof has endonuclease activity; and a guide RNA, or comprises a nucleic acid encoding the guide RNA, and the guide RNA can bind to a specific nuclease.

[0015] According to an embodiment of the present application, a recombinant vector can be provided, wherein, the recombinant vector comprises the nucleic acid encoding the nuclease described in the present application, the guide RNA described in the present application, the nucleic acid described in the present application, the nucleic acid construct described in the present application, or the composition described in the present application.

[0016] According to an embodiment of the present application, a recombinant host cell can be provided, wherein, the recombinant host cell comprises the nuclease described in the present application, the guide RNA described in the present application, the nucleic acid described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, or the recombinant vector described in the present application.

[0017] According to an embodiment of the present application, a method for introducing a double-strand break into a targeting gene of a host cell can be provided, wherein the method comprises: delivering the nuclease described in the present application, the guide RNA described in the present application, the nucleic acid described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, or the recombinant vector described in the present application into a host cell.

[0018] According to an embodiment of the present application, a method for deleting, replacing or inserting a targeting gene of a host cell can be provided, wherein the method comprises: delivering the nuclease described in the present application, the guide RNA described in the present application, the nucleic acid described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, or the recombinant vector described in the present application into a host cell.

[0019] According to an embodiment of the present application, a method for obtaining a host cell in which a targeting gene is deleted, replaced or inserted can be provided, wherein the method comprises: delivering the nuclease described in the present application, the guide RNA described in the present application, the nucleic acid described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, or the recombinant vector described in the present application into a host cell.

[0020] According to an embodiment of the present application, the use of the nuclease described in the present application, the guide RNA described in the present application, the nucleic acid described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, the recombinant vector described in the present application, or the recombinant host cell described in the present application for introducing a double-strand break into a targeting gene of a host cell can be provided.

[0021] According to an embodiment of the present application, the use of the nuclease described in the present application, the guide RNA described in the present application, the nucleic acid described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, the recombinant vector described in the present application, or the recombinant host cell described in the present application for deleting, replacing or inserting a targeting gene of a host cell can be provided.

[0022] According to an embodiment of the present application, the use of the nuclease described in the present application, the guide RNA described in the present application, the nucleic acid described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, the recombinant vector described in the present application, or the recombinant host cell described in the present application for preparing a drug or a preparation for gene therapy, cell therapy, genome research, and stem cell induction and post-induction differentiation can be provided.

[0023] According to an embodiment of the present application, a kit can be provided, wherein, the kit comprises the nuclease described in the present application, the guide RNA described in the present application, the nucleic acid described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, the recombinant vector described in the present application, or the recombinant host cell described in the present application.

[0024] The protein molecular weight of the nuclease described in the present application is far less than that of spCas9, about less than one third of the latter, which provides more possibilities for a variety of in vivo delivery in subsequent gene therapy, and can solve the problem of having difficulty in the delivery of spCas9 caused by the protein size; and compared with asCas 12 which also has a low protein molecular weight, the nuclease has higher gene editing efficiency, which provides the possibility of same becoming a new gene editing application tool; additionally, since different nucleases can recognize different transposon-associated motifs, the novel nuclease discovered in the present application brings more choices for subsequent application scenarios of different scales.

[0025] It should be understood that the content described in this section is not intended to identify critical or important features of the examples of the present application and is not used to limit the scope of the present application. Other features of the present application will be easily understood through the following description.BRIEF DESCRIPTION OF THE DRAWINGS

[0026] The accompanying drawings exemplarily show embodiments and form a part of the specification, and are used to explain exemplary implementations of the embodiments together with a written description of the specification. The embodiments shown are merely for illustrative purposes and do not limit the scope of the claims. Throughout the accompanying drawings, the same reference numerals denote similar but not necessarily same elements.

[0027] FIG. 1 shows a schematic diagram of an RGS dual fluorescence surrogate reporter system in example 1.

[0028] FIG. 2 shows flow cytometry plots with percentages of mRFP+eGFP+ cells presented at the top-right Q2 gate.

[0029] FIG. 3 shows GFP expression for all the active nuclease candidates of TP_A_1, TP_A_2, TP_A_8, TP_A_12, TP_A_18, TP_B_18, TP_B_41, TP_B_46, TP_B_70, TP_B_71, TP_B_72, TP_B_73, TP_C_23, TP_C_67, TP_C_70, TP_C_74, TP_D_1, TP_D_3, TP_D_4, TP_D_8, TP_D_17, TP_D_18, TP_D_23, TP_D_24, TP_D_25, TP_D_27, TP_D_30, TP_D_32, TP_D_40, TP_D_43, TP_D_51, TP_D_59, TP_D_61, TP_D_66, TP_D_67, TP_D_71, TP_D_72, TP_D_73, TP_E_2, TP_E_15, TP_E_17, TP_E_48, TP_F_56, TP_F_71, TP_F_77, TP_F_80, TP_F_83, TP_F_85, TP_G_14, TP_G_19, TP_G_20, TP_G_24, TP_G_43, TP_G_52, TP_G_53, TP_G_61, TP_G_66, TP_G_72, TP_G_75, TP_G_83, TP_G_84, TP_H_1, TP_H_3, TP_H_4, TP_H_5, TP_H_6, TP_H_9, TP_H_11, TP_H_12, TP_H_13, TP_H_15, TP_H_18, TP_H_19, TP_H_20, TP_H_21, TP_H_23, TP_H_24, TP_H_30, TP_H_31, TP_H_32, TP_H_34, TP_H_38, TP_H_39, TP_H_40, TP_H_43, TP_I_1, TP_I_2, TP_I_3, TP_I_4, TP_I_5, TP_I_6, TP_I_7, TP_I_8, TP_I_9, TP_I_10, TP_I_11, TP_I_12, TP_I_13, TP_I_15, TP_I_16, TP_I_17, TP_I_18, TP_I_19, TP_I_20, TP_I_21, TP_I_22, TP_I_24, TP_I_25, TP_I_26, TP_I_29, TP_I_31, TP_I_35, TP_I_37, TP_I_38, TP_I_40, TP_I_41, TP_I_44, TP_I_45, TP_I_46, TP_I_47, TP_I_48, TP_I_49, TP_I_50, TP_I_51, TP_I_52, TP_I_53, TP_I_55, TP_I_56, TP_I_58, TP_I_59, TP_I_61, TP_I_62, TP_I_64, TP_I_65, TP_I_66, TP_I_67, TP_I_70, TP_I_71, TP_I_76, TP_I_77, TP_I_79, TP_I_80, TP_I_82, TP_I_84, TP_I_85, TP_I_86, TP_I_87, TP_L_1, TP_L_4, TP_L_5, TP_L_8, TP_L_9, TP_L_10, TP_L_11, TP_L_12, TP_L_15, TP_L_16, TP_L_17, TP_L_21, TP_L_22, TP_L_24, TP_L_25, TP_L_26, TP_L_27, TP_L_28, TP_L_31, TP_L_32, TP_L_34, TP_L_36, TP_L_37, TP_L_39, TP_M_1, TP_M_3, TP_M_7, TP_M_11, TP_M_14, TP_M_17, TP_M_19, TP_M_20, TP_M_24, TP_M_31, TP_M_32, TP_M_33, TP_M_34, TP_M_35, TP_M_37, TP_M_40, TP_M_41, TP_M_43, TP_M_46, TP_M_49, TP_M_58, TP_M_65, TP_M_66, TP_M_67, TP_M_70 and TP_M_78. All the results were quantified by flow cytometry assay as previous shown in example 2.

[0030] FIG. 4, FIG. 5, FIG. 6, FIG. 7 and FIG. 8 are the partial enlarged pictures of FIG. 3.

[0031] FIG. 9 shows endogenous editing efficiency (quantified by the proportion of reads with insertions or deletions at target site) of TP_C_23, TP_D_51, TP_D_67, TP_E_15, TP_F_85, TP_G_24, TP_H_5, TP_H_6, TP_H_9, TP_H_11, TP_H_24, TP_H_30, TP_H_32, TP_H_34, TP_H_38, TP_I_1, TP_I_5, TP_I_6, TP_I_12, TP_I_15, TP_I_18, TP_I_20, TP_I_38, TP_I_49, TP_I_64 and TP_I_79 in example 3.

[0032] FIG. 10 shows an evolutionary branching diagram of TP_A_1, TP_A_2, TP_A_8, TP_A_12, TP_A_18, TP_B_18, TP_B_41, TP_B_46, TP_B_70, TP_B_71, TP_B_72, TP_B_73, TP_C_23, TP_C_67, TP_C_70, TP_C_74, TP_D_1, TP_D_3, TP_D_4, TP_D_8, TP_D_17, TP_D_18, TP_D_23, TP_D_24, TP_D_25, TP_D_27, TP_D_30, TP_D_32, TP_D_40, TP_D_43, TP_D_51, TP_D_59, TP_D_61, TP_D_66, TP_D_67, TP_D_71, TP_D_72, TP_D_73, TP_E_2, TP_E_15, TP_E_17, TP_E_48, TP_F_56, TP_F_71, TP_F_77, TP_F_80, TP_F_83, TP_F_85, TP_G_14, TP_G_19, TP_G_20, TP_G_24, TP_G_43, TP_G_52, TP_G_53, TP_G_61, TP_G_66, TP_G_72, TP_G_75, TP_G_83, TP_G_84, TP_H_1, TP_H_3, TP_H_4, TP_H_5, TP_H_6, TP_H_9, TP_H_11, TP_H_12, TP_H_13, TP_H_15, TP_H_18, TP_H_19, TP_H_20, TP_H_21, TP_H_23, TP_H_24, TP_H_30, TP_H_31, TP_H_32, TP_H_34, TP_H_38, TP_H_39, TP_H_40, TP_H_43, TP_I_1, TP_I_2, TP_I_3, TP_I_4, TP_I_5, TP_I_6, TP_I_7, TP_I_8, TP_I_9, TP_I_10, TP_I_11, TP_I_12, TP_I_13, TP_I_15, TP_I_16, TP_I_17, TP_I_18, TP_I_19, TP_I_20, TP_I_21, TP_I_22, TP_I_24, TP_I_25, TP_I_26, TP_I_29, TP_I_31, TP_I_35, TP_I_37, TP_I_38, TP_I_40, TP_I_41, TP_I_44, TP_I_45, TP_I_46, TP_I_47, TP_I_48, TP_I_49, TP_I_50, TP_I_51, TP_I_52, TP_I_53, TP_I_55, TP_I_56, TP_I_58, TP_I_59, TP_I_61, TP_I_62, TP_I_64, TP_I_65, TP_I_66, TP_I_67, TP_I_70, TP_I_71, TP_I_76, TP_I_77, TP_I_79, TP_I_80, TP_I_82, TP_I_84, TP_I_85, TP_I_86, TP_I_87, TP_L_1, TP_L_4, TP_L_5, TP_L_8, TP_L_9, TP_L_10, TP_L_11, TP_L_12, TP_L_15, TP_L_16, TP_L_17, TP_L_21, TP_L_22, TP_L_24, TP_L_25, TP_L_26, TP_L_27, TP_L_28, TP_L_31, TP_L_32, TP_L_34, TP_L_36, TP_L_37, TP_L_39, TP_M_1, TP_M_3, TP_M_7, TP_M_11, TP_M_14, TP_M_17, TP_M_19, TP_M_20, TP_M_24, TP_M_31, TP_M_32, TP_M_33, TP_M_34, TP_M_35, TP_M_37, TP_M_40, TP_M_41, TP_M_43, TP_M_46, TP_M_49, TP_M_58, TP_M_65, TP_M_66, TP_M_67, TP_M_70, TP_M_78 and ISDra2 based on protein sequences in example 2.

[0033] FIG. 11 and FIG. 12 are the partial enlarged pictures of FIG. 10.

[0034] FIG. 13 shows a schematic diagram illustrating the order of elements in the report vectors in example 4. FIG. 14 shows a schematic diagram demonstrating how the reporter vectors work in example 4.

[0035] FIGS. 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, and 31 show YFP expression in example 4 for all the active nuclease candidates of TP_A_1, TP_A_2, TP_A_8, TP_A_12, TP_A_18, TP_B_18, TP_B_41, TP_B_46, TP_B_70, TP_B_71, TP_B_72, TP_B_73, TP_C_23, TP_C_67, TP_C_70, TP_C_74, TP_D_1, TP_D_3, TP_D_4, TP_D_8, TP_D_17, TP_D_18, TP_D_23, TP_D_24, TP_D_25, TP_D_27, TP_D_30, TP_D_32, TP_D_40, TP_D_43, TP_D_51, TP_D_59, TP_D_61, TP_D_66, TP_D_67, TP_D_71, TP_D_72, TP_D_73, TP_E_2, TP_E_15, TP_E_17, TP_E_48, TP_F_56, TP_F_71, TP_F_77, TP_F_80, TP_F_83, TP_F_85, TP_G_14, TP_G_19, TP_G_20, TP_G_24, TP_G_43, TP_G_52, TP_G_53, TP_G_61, TP_G_66, TP_G_72, TP_G_75, TP_G_83, TP_G_84, TP_H_1, TP_H_3, TP_H_4, TP_H_5, TP_H_6, TP_H_9, TP_H_11, TP_H_12, TP_H_13, TP_H_15, TP_H_18, TP_H_19, TP_H_20, TP_H_21, TP_H_23, TP_H_24, TP_H_30, TP_H_31, TP_H_32, TP_H_34, TP_H_38, TP_H_39, TP_H_40, TP_H_43, TP_I_1, TP_I_2, TP_I_3, TP_I_4, TP_I_5, TP_I_6, TP_I_7, TP_I_8, TP_I_9, TP_I_10, TP_I_11, TP_I_12, TP_I_13, TP_I_15, TP_I_16, TP_I_17, TP_I_18, TP_I_19, TP_I_20, TP_I_21, TP_I_22, TP_I_24, TP_I_25, TP_I_26, TP_I_29, TP_I_31, TP_I_35, TP_I_37, TP_I_38, TP_I_40, TP_I_41, TP_I_44, TP_I_45, TP_I_46, TP_I_47, TP_I_48, TP_I_49, TP_I_50, TP_I_51, TP_I_52, TP_I_53, TP_I_55, TP_I_56, TP_I_58, TP_I_59, TP_I_61, TP_I_62, TP_I_64, TP_I_65, TP_I_66, TP_I_67, TP_I_70, TP_I_71, TP_I_76, TP_I_77, TP_I_79, TP_I_80, TP_I_82, TP_I_84, TP_I_85, TP_I_86, TP_I_87, TP_L_1, TP_L_4, TP_L_5, TP_L_8, TP_L_9, TP_L_10, TP_L_11, TP_L_12, TP_L_15, TP_L_16, TP_L_17, TP_L_21, TP_L_22, TP_L_24, TP_L_25, TP_L_26, TP_L_27, TP_L_28, TP_L_31, TP_L_32, TP_L_34, TP_L_36, TP_L_37, TP_L_39, TP_M_1, TP_M_3, TP_M_7, TP_M_11, TP_M_14, TP_M_17, TP_M_19, TP_M_20, TP_M_24, TP_M_31, TP_M_32, TP_M_33, TP_M_34, TP_M_35, TP_M_37, TP_M_40, TP_M_41, TP_M_43, TP_M_46, TP_M_49, TP_M_58, TP_M_65, TP_M_66, TP_M_67, TP_M_70 and TP_M_78.DETAILED DESCRIPTION OF EMBODIMENTS

[0036] Unless otherwise indicated or contradicts the context, the terms or expressions used herein should be read in conjunction with the entire content of the present disclosure and as understood by those of ordinary skill in the art. All technical and scientific terms used herein have the same meanings as commonly understood by those of ordinary skill in the art, unless otherwise defined.

[0037] In the present application, the terms “nucleic acid” and “polynucleotide” are used interchangeably, and refer to polymerization forms of nucleotides of any length, including deoxyribonucleotides, ribonucleotides, combinations thereof, and analogs thereof.

[0038] In the present application, the terms “polypeptide” and “peptide” are used interchangeably, and refer to polymers of amino acids of any length. Therefore, polypeptides, oligopeptides, proteins, antibodies and enzymes are all included in the definition of polypeptide.

[0039] As described in the present application, the “fragment” of a sequence refers to a portion of a sequence. For example, the fragment of a nucleic acid sequence refers to a portion of the nucleic acid sequence, and the fragment of an amino acid sequence refers to a portion of the amino acid sequence.

[0040] As described in the present application, a “variant” of a sequence is a polynucleotide or polypeptide that differs from a reference polynucleotide or polypeptide, respectively, but retains essential properties. A typical variant of a polynucleotide differs in nucleic acid sequence from another reference polynucleotide, and the differences in nucleic acid sequence may or may not alter the amino acid sequence of the polypeptide encoded by the reference polynucleotide. A typical variant of a polypeptide differs in amino acid sequence from another reference polypeptide. Generally, the differences are limited so that the sequences of the reference polypeptide and the variant are generally very similar, and are identical in many regions. A variant polypeptide and a reference polypeptide may differ in amino acid sequence by one or more substitutions, additions, deletions in any combination. The substituted or inserted amino acid residue may or may not be a residue encoded by the genetic code. Variants of polynucleotides or polypeptides may be naturally occurring, such as allelic variations, or they may be unknown naturally occurring variants. Non-naturally occurring polynucleotide and polypeptide variants can be produced by mutagenesis techniques, direct synthesis, and other recombinant methods known to the skilled artisan.

[0041] Amino acids are usually classified by the properties of their side chains. For example, side chains may render amino acids weakacids (e.g., amino acids D and E) or weak bases (e.g., amino acids K, R and H); and if the side chains are polar, the amino acids become hydrophilic (e.g., amino acids L and I), or if the side chains are nonpolar, the amino acids become hydrophobic (e.g., amino acids S and C).

[0042] As described in the present application, the “aliphatic amino acid” has a side chain that is an aliphatic group. Aliphatic groups cause amino acids to be nonpolar and hydrophobic. The aliphatic group is preferably an unsubstituted branched or linear alkyl group. Non-limiting examples of the aliphatic amino acids are A (alanine), V (valine), L (leucine), I (isoleucine), M (methionine), D (aspartic acid), E (glutamic acid), K (lysine), R (arginine), G (glycine), S (serine), T (threonine), C (cysteine), N (asparagine), and Q (glutamine).

[0043] As described in the present application, the “nonpolar amino acid” has a nonpolar side chain that makes the amino acid hydrophobic. Non-limiting examples of the nonpolar amino acid are A (alanine), V (valine), L (leucine), I (isoleucine), F (phenylalanine), W (tryptophan), M (methionine), P (proline), and G (glycine).

[0044] As described in the present application, the “polar amino acid” has a polar side chain that makes the amino acid hydrophilic. Non-limiting examples of the polar amino acid are T (threonine), S (serine), C (cysteine), N (asparagine), Q (glutamine), Y (tyrosine), K (lysine), R (arginine), H (histidine), D (aspartic acid), and E (glutamic acid). Polar amino acids can be divided into polar uncharged amino acids or polar charged amino acids.

[0045] As described in the present application, the “polar uncharged amino acid” has a polar side chain of uncharged residues. Non-limiting examples of the polar uncharged amino acid are T (threonine), S (serine), C (cysteine), N (asparagine), Q (glutamine), and Y (tyrosine).

[0046] As described in the present application, the “polar charged amino acid” has a polar side chain of at least one charged residue. Non-limiting examples of the polar charged amino acid are K (lysine), R (arginine), H (histidine), D (aspartic acid), and E (glutamic acid). Polar charged amino acids can be divided into positively charged amino acids or negatively charged amino acids.

[0047] As described in the present application, the “positively charged amino acid” has a polar side chain of at least one positively charged residue. Non-limiting examples of the positively charged amino acid are K (lysine), R (arginine), and H (histidine).

[0048] As described in the present application, the “negatively charged amino acid” has a polar side chain of at least one negatively charged residue. Non-limiting examples of the negatively charged amino acid are D (aspartic acid), and E (glutamic acid).

[0049] The term “family” as used in the present application refers to a group of nucleic acids or proteins having high structural similarity produced by the same ancestor by means of replication and variation, which usually have related or even the same functions.

[0050] The term “nuclease” described in the present application refers to an enzyme capable of cleaving phosphodiester bonds. Nucleases hydrolyze the phosphodiester bonds in the backbone of nucleic acids. The term “endonuclease” described in the present application refers to an enzyme capable of cleaving phosphodiester bonds between nucleotides.

[0051] The term “guide RNA” described in the present application refers to any RNA molecule that can form a complex with the nuclease described in the present application. For example, the guide RNA can be a molecule that recognizes a targeting gene. In some embodiments of the present application, the guide RNA comprises a reRNA and a targeted sequence, wherein the reRNA can bind to a particular nuclease, and the targeted sequence can be designed to be complementary to a target strand of a targeting gene.

[0052] The term “transposon-associated motif” (TAM) described in the present application refers to a short nucleotide sequence adjacent to a targeting gene, which sequence can be recognized by a complex formed by nuclease and guide RNA described in the present application. If a targeting gene is not adjacent to a transposon-associated motif, the nuclease cannot successfully recognize the targeting gene. Sequences and lengths of the transposon-associated motif in the present application can vary depending on the nuclease.

[0053] The terms “targeting gene”“targeting sequence”“targeting nucleic acid”“gene of interest”, “sequence of interest” and “nucleic acid of interest” described in the present application are used interchangeably, and refer to nucleotide sequences on chromosomal DNA, chloroplast DNA, mitochondrial DNA, plasmid DNA, or any other DNA molecule in the genome of cells, which sequences can be recognized, bound to, and selectively cleaved by a complex formed by the nuclease and guide RNA described in the present application.

[0054] The term “nucleic acid construct” as used in the present application is defined as a single-stranded or double-stranded nucleic acid molecule herein, and preferably refers to an artificially constructed nucleic acid molecule. Optionally, the nucleic acid construct further includes one or more operably linked regulatory sequences, which can direct the expression of a coding sequence in a suitable host cell under compatible conditions. The term “expression” is understood to include any step involved in the production of a protein or polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification and secretion. The term “regulatory sequence” includes all components necessary or advantageous for expression of the polypeptide / protein of the present application. Each regulatory sequence may be naturally present or exogenous to the nucleic acid sequence encoding the protein or polypeptide. These regulatory sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal sequences, and transcription terminators. At a minimum, the regulatory sequences should include promoters and termination signals for transcription and translation. Regulatory sequences with linkers can be provided for the purpose of introduction into specific restriction sites for linking the regulatory sequences to the coding region of a nucleic acid sequence encoding a protein or polypeptide.

[0055] The term “promoter” as used in the present application refers to a polynucleotide sequence that can control the transcription of a coding sequence. Promoter sequences include specific sequences sufficient to enable RNA polymerase to recognize, bind, and initiate transcription. In addition, promoter sequences may include sequences that optionally modulate the recognition, binding and transcription initiation activities of RNA polymerase in the nucleic acid construct provided in the present application. A promoter can affect the transcription of a gene located on the same nucleic acid molecule as the promoter or a gene located on a different nucleic acid molecule from the promoter.

[0056] The term “host cell” as used in the present application include, but are not limited to, an animal cell, a plant cell, an algal cell, a fungal cell, a yeast cell, or a bacterial cell. This term includes a progeny of an original cell into which an exogenous nucleic acid fragment has been introduced. Exemplary host cell includes human embryonic kidney cell HEK293T. It is understood that, due to natural, accidental or intentional mutations, the progeny of a single parent cell may not necessarily be identical to the original parent morphologically or in terms of genome or total DNA complement.

[0057] The term “vector” as used in the present application refers to a nucleic acid molecule capable of transporting another nucleic acid molecule connected to it. Examples of vectors include, but are not limited to, plasmids, viruses, bacteria, phages, and insertable DNAfragments. The term “plasmid” refers to a circular double-stranded DNA capable of accepting an exogenous nucleic acid fragment and replicating in prokaryotic or eukaryotic cells.Nuclease

[0058] The present application provides an isolated nuclease, wherein the nuclease comprises an amino acid sequence as shown in the following formula:(X1)(X2)a(X3)(X4)(X5)b(X6)(X7)c(X8)(X9)d(X10)(X11)e(X12)(X13)f(X14)(X15)(X16)g wherein a, b, c, d, e, f, and g are the numbers of amino acids; (X1), (X3), (X4), (X6), (X8), (X10), (X12), (X14), and (X16) are independently polar amino acids or aliphatic amino acids; (X2) is any amino acid, and a is 15 or 16; (X5) is any amino acid, and b is 2; (X7) is any amino acid, and c is 2, 3 or 4; (X9) is any amino acid, and d is 14, 15, 16, 17 or 18; (X11) is any amino acid, and e is 1 or 2; (X13) is any amino acid, and f is 6; and (X15) is any amino acid, and g is 5.

[0060] In some embodiments, the (X1) is a positively charged amino acid; (X3) is a polar uncharged amino acid; (X4) is a polar uncharged amino acid; (X6) is a polar uncharged amino acid; (X8) is a polar uncharged amino acid; (X10) is a polar uncharged amino acid; (X12) is a polar uncharged amino acid; (X14) is a negatively charged amino acid; and (X16) is a polar uncharged amino acid. In some embodiments, the (X1) is K. In some embodiments, the (X3) is S or T. In some embodiments, the (X4) is S or T. In some embodiments, the (X6) is C. In some embodiments, the (X8) is C. In some embodiments, the (X10) is C. In some embodiments, the (X12) is C. In some embodiments, the (X14) is D. In some embodiments, the (X16) is N.

[0061] According to an embodiment of the present application, an isolated nuclease can be provided, wherein the nuclease has a nuclease sequence selected from the following (i) or a variant sequence of the aforementioned nuclease having a nuclease activity in (ii)-(iv): (i) at least one amino acid sequence as shown in any one of SEQ ID NOs: 1-197; (ii) at least one of sequences obtained by performing deletion, substitution, insertion, or mutation of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids on the amino acid sequence as shown in any one of SEQ ID NOs: 1-197; (iii) at least one of amino acid sequences having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence as shown in any one of SEQ ID NOs: 1-197; and (iv) at least one of sequences obtained by further fusing the amino acid sequence as shown in any one of SEQ ID NOs: 1-197 with other sequences.

[0062] In some embodiments, the nuclease has a nuclease sequence selected from at least one of the following groups (1)-(9): (1) at least one amino acid sequence as shown in any one of SEQ ID NOs: 52 and 113-147; (2) at least one amino acid sequence as shown in any one of SEQ ID NOs: 27-28, 36-38, 62-85 and 148-171; (3) at least one amino acid sequence as shown in any one of SEQ ID NOs: 13, 86-100 and 105-110; (4) at least one amino acid sequence as shown in any one of SEQ ID NOs: 10-11, 17-19, 29-30 and 174-180; (5) at least one amino acid sequence as shown in any one of SEQ ID NOs: 34, 35, 50, 61 and 181-189; (6) at least one amino acid sequence as shown in any one of SEQ ID NOs: 53 and 190-197; (7) at least one amino acid sequence as shown in any one of SEQ ID NOs: 101, 103, 104 and 112; (8) at least one amino acid sequence as shown in any one of SEQ ID NOs: 7 and 23-25; and (9) at least one amino acid sequence as shown in any one of SEQ ID NOs: 3, 21 and 22.

[0063] In some embodiments, the nuclease has a nuclease sequence selected from at least one of the following groups (1)-(12): (1) at least one amino acid sequence as shown in any one of SEQ ID NOs: 1, 3-4, 6-7, 21-23, 50, 52, 60-61 and 113-147; (2) at least one amino acid sequence as shown in any one of SEQ ID NOs: 14, 27-28, 36-38, 45-48, 59, 62-85 and 148-171; (3) at least one amino acid sequence as shown in any one of SEQ ID NOs: 13, 43 and 86-112; (4) at least one amino acid sequence as shown in any one of SEQ ID NOs: 15-16, 24-25, 32-35 and 181-189; (5) at least one amino acid sequence as shown in any one of SEQ ID NOs: 9, 11, 17-19, 29 and 174-180; (6) at least one amino acid sequence as shown in any one of SEQ ID NOs: 10, 12, 26, 30, 42 and 58; (7) at least one amino acid sequence as shown in any one of SEQ ID NOs: 2, 20 and 31; (8) at least one amino acid sequence as shown in any one of SEQ ID NOs: 8 and 51; (9) at least one amino acid sequence as shown in any one of SEQ ID NOs: 39 and 49; (10) at least one amino acid sequence as shown in any one of SEQ ID NOs: 54 and 55; (11) at least one amino acid sequence as shown in any one of SEQ ID NOs: 53 and 190-197; and (12) at least one amino acid sequence as shown in any one of SEQ ID NOs: 5, 172 and 173.

[0064] In some embodiments, the nuclease belongs to the IS200 / IS605 family. In some embodiments, the nuclease belongs to the IS605, or IS1341 subfamily. In some embodiments, the species sources of the nuclease include Bacteria or Archaea. In some embodiments, the species sources of the nuclease include Actinobacteria, Aquificae, Bacteroidetes, Candidatus Poribacteria, Chloroflexi, Cyanobacteria, Deinococcusthermus, Firmicutes, Planctomycetes, Proteobacteria, Spirochaetes, Tenericutes, Thermotogae, Verrucomicrobia, Candidatus Micrarchaeota, Crenarchaeota, or Euryarchaeota. Guide RNA

[0065] According to an embodiment of the present application, a guide RNA can be provided, wherein the guide RNA comprises a reRNA, the reRNA comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 198-394 or a variant thereof, and the guide RNA can bind to a specific nuclease. In some embodiments, the reRNA comprises at least one of nucleotide sequences having at least 70%, 80%, 90%, 95% or 99% identity to the nucleotide sequence as shown in any one of SEQ ID NOs: 198-394. In some embodiments, the reRNA comprises at least one of the nucleotide sequences as shown in any one of SEQ ID NOs: 198-394. In some embodiments, the reRNA is at least one of the nucleotide sequences as shown in any one of SEQ ID NOs: 198-394.

[0066] In some embodiments, the guide RNA further comprises a targeted sequence that can recognize a targeting gene adjacent to a transposon-associated motif. In some embodiments, the targeted sequence is of at least one of 10-50, 10-40, 10-30, or 15-25 nucleotides in length.

[0067] Sequences and lengths of the transposon-associated motif in the present application can vary depending on the nuclease, and the transposon-associated motif can be recognized by a complex formed by the nuclease and guide RNA described in the present application. In some embodiments, the transposon-associated motif comprises a nucleotide sequence as shown in the following formula:(X17)h(X18)(X19)A(X20)wherein h is the number of nucleotides; A is an adenine deoxyribonucleotide; (X17) is any deoxyribonucleotide, and h is 0 or 1; (X18) is a cytosine deoxyribonucleotide or thymine deoxyribonucleotide; (X19) is a cytosine deoxyribonucleotide, thymine deoxyribonucleotide, or guanine deoxyribonucleotide; and (X20) is any deoxyribonucleotide.Nucleicacid, Nucleicacid Construct

[0069] According to an embodiment of the present application, a nucleic acid can be provided, wherein, the nucleic acid encodes the nuclease described in the present application and / or the guide RNA described in the present application.

[0070] According to an embodiment of the present application, a nucleic acid construct can be provided, comprising the nucleic acid described in the present application. In some embodiments, the nucleic acid construct further comprising a promoter. The promoter can be any suitable promoter sequence, that is, a nucleic acid sequence that can be recognized by a host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates the expression of the protein or polypeptide. The promoter can be any nucleic acid sequence having transcriptional activity in a selected host cell, including mutant, truncated and heterozygous promoters, and can be derived from genes encoding extracellular or intracellular proteins or polypeptides homologous or heterologous to the host cell. In some embodiments, the promoter includes CMV, EF1a, SV40, PGK, UbC, human beta actin, CAG, TRE, UAS, Ac5, GFAP, Polyhedrin promotor, TBG, ALB, ApoEHCR-hAAT, CaMKIIa, GAL1, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6, T7, T7lac, Sp6, araBAD, trp, lac, Ptac, or pL.

[0071] In some embodiments, the nucleic acid construct is modified by 5′-end capping and / or 3′-end polyadenylating, and the nucleic acid construct retains the activity of nuclease and / or guide RNA. In some embodiments, the nucleic acid construct is modified by thiophosphate bond modification, 2′-MOE (2-O-(2-methoxyethyl)), PNA (peptide nucleic acid), GNA (glycerol nucleic acid), LNA (locked nucleic acid), GalNAc (N-acetylgalactosamine) LNP (lipid nano particle) PNP (peptide nanoparticles). The modification methods of nucleic acid are known in the art, the entire contents of which are hereby incorporated by reference.

[0072] In some embodiments, the nucleic acid construct further comprises a poly A sequence. Poly Atailing signal sequences well known in the art, as well as various truncated forms of polyA tailing signals, can be used in the present application.

[0073] In some embodiments, the nucleic acid construct further includes any transcription termination sequence, i.e., a sequence that is recognized by the host cell to terminate transcription. The termination sequence is operably linked to the 3′-terminus of the nucleic acid sequence encoding the protein or polypeptide. Any terminator that is functional in the host cell of choice can be used in the present invention.

[0074] Optionally, the nucleic acid construct may further include a suitable leader sequence, that is, an untranslated region in the mRNA that is important for translation in the host cell. The leader sequence is operably linked to the 5′-terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice can be used in the present invention.

[0075] Optionally, the nucleic acid construct may further include a propeptide coding region, which encodes an amino acid sequence located at the amino terminus of the polypeptide. The resulting polypeptide is called a zymogen or a propolypeptide. The propolypeptide is usually inactive and can be converted into a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.

[0076] Optionally, the nucleic acid construct may further include a regulatory sequence that can regulate the expression of the polypeptide according to the growth conditions of the host cell. Examples of the regulatory sequence are systems that turn gene expression on or off in response to chemical or physical stimuli, including in the presence of regulatory compounds. Other examples of the regulatory sequence are those that enable gene amplification. In these instances, the nucleic acid sequence encoding the protein or polypeptide should be operably linked to the regulatory sequence.Composition

[0077] According to an embodiment of the present application, a composition may be provided, wherein, the composition includes: an IS200 / IS605 family nuclease or a functional fragment thereof, or comprises a nucleic acid encoding the IS200 / IS605 family nuclease or the functional fragment thereof, and the nuclease or the functional fragment thereof has endonuclease activity; and a guide RNA, or comprises a nucleic acid encoding the guide RNA, and the guide RNA can bind to a specific nuclease.

[0078] In some embodiments, the composition is selected from at least one of the following groups (1)-(198), and any one of the following groups (1)-(198) comprises: a nuclease-related sequence and a guide RNA-related sequence,

[0079] (1) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 1 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 198;

[0080] (2) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 2 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 199;

[0081] (3) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 3 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 200;

[0082] (4) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 4 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 201;

[0083] (5) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 5 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 202;

[0084] (6) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 6 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 203;

[0085] (7) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 7 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 204;

[0086] (8) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 8 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 205;

[0087] (9) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 9 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 206;

[0088] (10) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 10 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 207;

[0089] (11) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 11 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 208;

[0090] (12) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 12 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 209;

[0091] (13) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 13 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 210;

[0092] (14) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 14 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 211;

[0093] (15) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 15 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 212;

[0094] (16) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 16 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 213;

[0095] (17) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 17 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 214;

[0096] (18) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 18 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 215;

[0097] (19) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 19 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 216;

[0098] (20) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 20 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 217;

[0099] (21) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 21 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 218;

[0100] (22) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 22 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 219;

[0101] (23) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 23 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 220;

[0102] (24) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 24 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 221;

[0103] (25) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 25 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 222;

[0104] (26) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 26 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 223;

[0105] (27) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 27 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 224;

[0106] (28) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 28 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 225;

[0107] (29) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 29 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 226;

[0108] (30) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 30 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 227;

[0109] (31) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 31 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 228;

[0110] (32) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 32 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 229;

[0111] (33) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 33 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 230;

[0112] (34) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 34 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 231;

[0113] (35) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 35 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 232;

[0114] (36) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 36 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 233;

[0115] (37) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 37 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 234;

[0116] (38) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 38 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 235;

[0117] (39) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 39 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 236;

[0118] (40) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 40 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 237;

[0119] (41) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 41 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 238;

[0120] (42) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 42 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 239;

[0121] (43) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 43 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 240;

[0122] (44) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 44 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 241;

[0123] (45) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 45 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 242;

[0124] (46) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 46 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 243;

[0125] (47) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 47 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 244;

[0126] (48) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 48 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 245;

[0127] (49) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 49 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 246;

[0128] (50) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 50 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 247;

[0129] (51) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 51 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 248;

[0130] (52) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 52 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 249;

[0131] (53) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 53 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 250;

[0132] (54) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 54 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 251;

[0133] (55) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 55 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 252;

[0134] (56) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 56 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 253;

[0135] (57) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 57 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 254;

[0136] (58) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 58 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 255;

[0137] (59) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 59 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 256;

[0138] (60) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 60 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 257;

[0139] (61) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 61 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 258;

[0140] (62) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 62 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 259;

[0141] (63) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 63 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 260;

[0142] (64) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 64 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 261;

[0143] (65) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 65 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 262;

[0144] (66) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 66 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 263;

[0145] (67) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 67 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 264;

[0146] (68) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 68 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 265;

[0147] (69) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 69 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 266;

[0148] (70) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 70 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 267;

[0149] (71) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 71 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 268;

[0150] (72) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 72 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 269;

[0151] (73) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 73 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 270;

[0152] (74) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 74 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 271;

[0153] (75) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 75 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 272;

[0154] (76) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 76 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 273;

[0155] (77) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 77 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 274;

[0156] (78) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 78 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 275;

[0157] (79) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 79 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 276;

[0158] (80) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 80 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 277;

[0159] (81) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 81 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 278;

[0160] (82) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 82 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 279;

[0161] (83) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 83 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 280;

[0162] (84) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 84 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 281;

[0163] (85) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 85 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 282;

[0164] (86) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 86 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 283;

[0165] (87) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 87 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 284;

[0166] (88) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 88 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 285;

[0167] (89) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 89 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 286;

[0168] (90) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 90 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 287;

[0169] (91) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 91 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 288;

[0170] (92) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 92 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 289;

[0171] (93) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 93 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 290;

[0172] (94) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 94 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 291;

[0173] (95) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 95 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 292;

[0174] (96) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 96 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 293;

[0175] (97) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 97 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 294;

[0176] (98) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 98 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 295;

[0177] (99) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 99 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 296;

[0178] (100) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 100 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 297;

[0179] (101) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 101 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 298;

[0180] (102) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 102 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 299;

[0181] (103) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 103 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 300;

[0182] (104) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 104 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 301;

[0183] (105) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 105 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 302;

[0184] (106) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 106 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 303;

[0185] (107) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 107 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 304;

[0186] (108) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 108 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 305;

[0187] (109) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 109 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 306;

[0188] (110) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 110 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 307;

[0189] (111) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 111 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 308;

[0190] (112) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 112 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 309;

[0191] (113) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 113 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 310;

[0192] (114) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 114 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 311;

[0193] (115) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 115 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 312;

[0194] (116) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 116 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 313;

[0195] (117) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 117 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 314;

[0196] (118) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 118 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 315;

[0197] (119) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 119 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 316;

[0198] (120) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 120 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 317;

[0199] (121) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 121 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 318;

[0200] (122) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 122 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 319;

[0201] (123) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 123 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 320;

[0202] (124) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 124 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 321;

[0203] (125) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 125 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 322;

[0204] (126) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 126 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 323;

[0205] (127) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 127 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 324;

[0206] (128) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 128 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 325;

[0207] (129) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 129 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 326;

[0208] (130) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 130 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 327;

[0209] (131) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 131 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 328;

[0210] (132) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 132 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 329;

[0211] (133) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 133 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 330;

[0212] (134) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 134 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 331;

[0213] (135) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 135 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 332;

[0214] (136) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 136 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 333;

[0215] (137) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 137 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 334;

[0216] (138) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 138 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 335;

[0217] (139) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 139 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 336;

[0218] (140) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 140 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 337;

[0219] (141) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 141 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 338;

[0220] (142) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 142 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 339;

[0221] (143) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 143 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 340;

[0222] (144) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 144 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 341;

[0223] (145) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 145 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 342;

[0224] (146) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 146 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 343;

[0225] (147) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 147 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 344;

[0226] (148) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 148 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 345;

[0227] (149) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 149 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 346;

[0228] (150) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 150 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 347;

[0229] (151) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 151 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 348;

[0230] (152) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 152 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 349;

[0231] (153) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 153 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 350;

[0232] (154) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 154 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 351;

[0233] (155) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 155 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 352;

[0234] (156) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 156 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 353;

[0235] (157) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 157 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 354;

[0236] (158) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 158 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 355;

[0237] (159) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 159 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 356;

[0238] (160) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 160 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 357;

[0239] (161) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 161 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 358;

[0240] (162) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 162 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 359;

[0241] (163) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 163 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 360;

[0242] (164) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 164 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 361;

[0243] (165) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 165 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 362;

[0244] (166) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 166 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 363;

[0245] (167) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 167 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 364;

[0246] (168) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 168 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 365;

[0247] (169) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 169 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 366;

[0248] (170) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 170 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 367;

[0249] (171) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 171 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 368;

[0250] (172) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 172 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 369;

[0251] (173) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 173 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 370;

[0252] (174) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 174 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 371;

[0253] (175) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 175 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 372;

[0254] (176) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 176 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 373;

[0255] (177) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 177 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 374;

[0256] (178) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 178 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 375;

[0257] (179) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 179 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 376;

[0258] (180) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 180 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 377;

[0259] (181) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 181 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 378;

[0260] (182) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 182 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 379;

[0261] (183) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 183 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 380;

[0262] (184) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 184 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 381;

[0263] (185) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 185 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 382;

[0264] (186) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 186 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 383;

[0265] (187) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 187 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 384;

[0266] (188) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 188 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 385;

[0267] (189) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 189 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 386;

[0268] (190) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 190 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 387;

[0269] (191) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 191 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 388;

[0270] (192) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 192 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 389;

[0271] (193) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 193 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 390;

[0272] (194) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 194 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 391;

[0273] (195) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 195 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 392;

[0274] (196) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 196 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 393;

[0275] (197) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 197 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 394;

[0276] (198) a variant of any one of the aforementioned groups (1)-(197),

[0277] wherein the nuclease-related sequence is the amino acid sequence of the variant of the nuclease in each group or a nucleic acid sequence encoding the variant, and the variant has a variant sequence of the aforementioned nuclease having a nuclease activity selected from the following (i)-(iii):

[0278] (i) at least one of sequences obtained by performing deletion, substitution, insertion, or mutation of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids on the amino acid sequence of the nuclease in each group;

[0279] (ii) at least one of amino acid sequences having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence as shown in any one of SEQ ID NOs: 1-197; and

[0280] (iii) at least one sequence obtained by further fusing the amino acid sequence as shown in any one of SEQ ID NO: 1-197 with other sequences.

[0281] In some embodiments, the guide RNA-related sequence further comprises a targeted sequence that can recognize a targeting gene adjacent to a transposon-associated motif. In some embodiments, the targeted sequence is of at least one of 10-50, 10-40, 10-30, or 15-25 nucleotides in length.

[0282] Sequences and lengths of the transposon-associated motif in the present application can vary depending on the nuclease, and the transposon-associated motif can be recognized by a complex formed by the nuclease and guide RNA described in the present application. In some embodiments, the transposon-associated motif comprises a nucleotide sequence as shown in the following formula:(X17)h(X18)(X19)A(X20)wherein h is the number of nucleotides; A is an adenine deoxyribonucleotide; (X17) is any deoxyribonucleotide, and h is 0 or 1; (X18) is a cytosine deoxyribonucleotid e or thymine deoxyribonucleotide; (X19) is a cytosine deoxyribonucleotide, thymine deoxyribonucleotide, or guanine deoxyribonucleotide; and (X20) is any deoxyribonucleotide.

[0284] The targeting gene in the present application includes any gene of interest, e.g., a gene of a natural functional protein, an artificial chimeric gene, or a gene of a non-coding RNA. In some embodiments, the gene of a natural functional protein includes a fluorescein reporter gene, a luciferase gene, and a resistance gene. In some embodiments, the artificial chimeric gene includes a gene of a chimeric antigen receptor. In some embodiments, the fluorescein reporter gene includes a gene encoding a green fluorescent protein, a red fluorescent protein, a blue fluorescent protein, or a yellow fluorescent protein. In some embodiments, the luciferase gene includes a gene encoding firefly luciferase or sea kidney luciferase. In some embodiments, the resistance gene includes a gene encoding puromycin resistance, G418 resistance, kanamycin resistance, tetracycline resistance, or bleomycin resistance.

[0285] In some embodiments, the nucleic acid encoding the amino acid sequence and / or the nucleic acid encoding the guide RNA further comprises a promoter. The promoter can be any suitable promoter sequence, that is, a nucleic acid sequence that can be recognized by a host cell expressing the nucleic acid sequence. The promoter sequence contains a transcriptional regulatory sequence that mediates the expression of the protein or polypeptide. The promoter can be any nucleic acid sequence having transcriptional activity in a selected host cell, including mutant, truncated and heterozygous promoters, and can be derived from genes encoding extracellular or intracellular proteins or polypeptides homologous or heterologous to the host cell. In some embodiments, the promoter includes CMV, EF1a, SV40, PGK, UbC, human beta actin, CAG, TRE, UAS, Ac5, GFAP, Polyhedrin promotor, TBG, ALB, ApoEHCR-hAAT, CaMKIIa, GAL1, TEF1, GDS, ADH1, CaMV35S, Ubi, H1, U6, T7, T7lac, Sp6, araBAD, trp, lac, Ptac, or pL. In some embodiments, the nucleic acid encoding the amino acid sequence and / or the nucleic acid encoding the guide RNA further comprises a polyA sequence. PolyA tailing signal sequences well known in the art, as well as various truncated forms of polyA tailing signals, can be used in the present application.

[0286] In some embodiments, the nucleic acid encoding the amino acid sequence and / or the nucleic acid encoding the guide RNA further comprises any transcription termination sequence that controls the expression of the exogenous nucleic acid fragment, i.e., a sequence that is recognized by a host cell to terminate transcription. Any terminator that is functional in the host cell of choice can be used in the present invention.

[0287] In some embodiments, the nucleic acid encoding the amino acid sequence and / or the nucleic acid encoding the guide RNA further comprises any transcription termination sequence, i.e., a sequence that is recognized by a host cell to terminate transcription. The termination sequence is operably linked to the 3′-terminus of the nucleic acid sequence encoding the protein or polypeptide. Any terminator that is functional in the host cell of choice can be used in the present invention.

[0288] Optionally, the nucleic acid encoding the amino acid sequence and / or the nucleic acid encoding the guide RNA may further comprise a suitable leader sequence, i.e., an untranslated region in the mRNA that is important for translation in the host cell. The leader sequence is operably linked to the 5′-terminus of the nucleic acid sequence encoding the polypeptide. Any leader sequence that is functional in the host cell of choice can be used in the present invention.

[0289] Optionally, the nucleic acid encoding the amino acid sequence and / or the nucleic acid encoding the guide RNA may further comprise a propeptide coding region, which encodes an amino acid sequence located at the amino terminus of the polypeptide. The resulting polypeptide is called a zymogen or a propolypeptide. The propolypeptide is usually inactive and can be converted into a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.

[0290] Optionally, the nucleic acid encoding the amino acid sequence and / or the nucleic acid encoding the guide RNA may further comprise a regulatory sequence that can regulate the expression of the polypeptide according to the growth conditions of the host cell. Examples of the regulatory sequence are systems that turn gene expression on or off in response to chemical or physical stimuli, including in the presence of regulatory compounds. Other examples of the regulatory sequence are those that enable gene amplification. In these instances, the nucleic acid sequence encoding the protein or polypeptide should be operably linked to the regulatory sequence.Recombinant Vector, Recombinant Host Cell and Kit

[0291] According to an embodiment of the present application, a recombinant vector can be provided, wherein, the recombinant vector comprises the nucleic acid encoding the nuclease described in the present application, the guide RNA described in the present application, the nucleic acid described in the present application, the nucleic acid construct described in the present application, or the composition described in the present application. The recombinant vector can be any suitable vector. In some embodiments, the recombinant vector includes, but is not limited to, a recombinant cloning vector, a recombinant eukaryotic expression plasmid, or a recombinant viral vector. In some embodiments, the recombinant eukaryotic expression plasmid includes pcDNA3.1, pCMV, pUC18, pUC19, pUC57, pBAD, pET, pENTR, pGenlenti, or pAAV. In some embodiments, the recombinant virus vector includes a recombinant adenovirus vector, a recombinant adeno-associated virus vector, a recombinant retrovirus vector, a recombinant herpes simplex virus vector, or a recombinant vaccinia virus vector. The recombinant vector of the present invention can be constructed using methods well known in the art. For example, depending on the restriction sites contained in the backbone vector used, appropriate restriction sites can be added to both ends of the nucleic acid construct of the present invention, and then loaded into the backbone vector.

[0292] According to an embodiment of the present application, a recombinant host cell can be provided, wherein, the recombinant host cell comprises the nuclease described in the present application, the guide RNA described in the present application, the nucleic acid described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, or the recombinant vector described in the present application. The recombinant host cell can be any host cell in which nucleases can be used. In some embodiments, the recombinant host cell includes, but is not limited to, an animal cell, a plant cell, an algal cell, a fungal cell, a yeast cell, or a bacterial cell. In some embodiments, the animal cell includes a mammalian cell. In some embodiments, the mammalian cell includes a primary cell (e.g., a mesenchymal stem cell, an endothelial cell, an epithelial cell, a fibroblast, a keratinocyte, a melanocyte, a smooth muscle cell, and an immune cell), an immortalized cell line (e.g., HEK293, NIH-3T3, RAW-264.7, STO, VERO, CT26, hTERT immortalized human endothelial / epithelial / fibroblast / keratinocyte / ductal / cell lines), a cancer cell line (e.g., Hela, HepG2 / 3, HL-60, HT-1080, HT-29, A549, SW620, HCT-15, HCT116, MDA-MB-231, MCF7, SK-OV-3, PANC-1, AsPc-1, THP-1, Huh7, KG-1, RAJI, HB-CB, Jurkat, K562, CRL5826, CHO, MDCK, and Renca), an embryonic stem cell line (e.g., H1, H9, WIBR2, WIBR3, G-Olig2, ESF158, RW. 4, R1, and D3) and differentiated cells thereof, or an induced pluripotent stem cell line and differentiated cells thereof. In some embodiments, the plant cell includes a monocot cell or a dicot cell. In some embodiments, the monocot cell or the dicot cell includes rice cell, maize cell, or soybean cell.

[0293] According to an embodiment of the present application, a kit can be provided, wherein, the kit comprises the nuclease described in the present application, the guide RNA described in the present application, the nucleic acid described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, the recombinant vector described in the present application, or the recombinant host cell described in the present application.Method and Use

[0294] The nuclease-based gene editing tools and methods provided in the present application can be applied to many fields such as gene therapy, molecular breeding in animals and plants, industrial microorganism engineering, model animal engineering, and scientific research. Particularly in the field of gene therapy, it can be applied for gene knockout based on DNA double-strand breaks in human genome.

[0295] According to an embodiment of the present application, a method for introducing a double-strand break into a targeting gene of a host cell can be provided, wherein the method comprises: delivering the nuclease described in the present application, the guide RNA described in the present application, the nucleic acid described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, or the recombinant vector described in the present application into a host cell.

[0296] According to an embodiment of the present application, a method for deleting, replacing or inserting a targeting gene of a host cell can be provided, wherein the method comprises: delivering the nuclease described in the present application, the guide RNA described in the present application, the nucleic acid described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, or the recombinant vector described in the present application into a host cell.

[0297] According to an embodiment of the present application, a method for obtaining a host cell in which a targeting gene is deleted, replaced or inserted can be provided, wherein the method comprises: delivering the nuclease described in the present application, the guide RNA described in the present application, the nucleic acid described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, or the recombinant vector described in the present application into a host cell.

[0298] The method of delivery into the host cell can be any suitable method. In some embodiments, the delivery method includes but is not limited to cationic liposome delivery, lipoid nanoparticulate delivery, cationic polymer delivery, vesicle-exosome delivery, gold nanoparticulate delivery, polypeptide and protein delivery, retrovirus delivery, lentivirus delivery, adenovirus delivery, adeno-associated virus delivery, electroporation, agrobacterium infection, or gene gun. The methods of cell transfection and culture are routine methods in the art, and appropriate transfection and culture methods can be selected according to different cell types.

[0299] The host cell can be any host cell in which nucleases can be used. In some embodiments, the host cell includes, but is not limited to, an animal cell, a plant cell, an algal cell, a fungal cell, a yeast cell, or a bacterial cell. In some embodiments, the animal cell includes a mammalian cell. In some embodiments, the mammalian cell includes a primary cell (e.g., a mesenchymal stem cell, an endothelial cell, an epithelial cell, a fibroblast, a keratinocyte, a melanocyte, a smooth muscle cell, and an immune cell), an immortalized cell line (e.g., HEK293, NIH-3T3, RAW-264.7, STO, VERO, CT26, hTERT immortalized human endothelial / epithelial / fibroblast / keratinocyte / ductal / cell lines), a cancer cell line (e.g., Hela, HepG2 / 3, HL-60, HT-1080, HT-29, A549, SW620, HCT-15, HCT116, MDA-MB-231, MCF7, SK-OV-3, PANC-1, AsPc-1, THP-1, Huh7, KG-1, RAJI, HB-CB, Jurkat, K562, CRL5826, CHO, MDCK, and Renca), an embryonic stem cell line (e.g., H1, H9, WIBR2, WIBR3, G-Olig2, ESF158, RW. 4, R1, and D3) and differentiated cells thereof, or an induced pluripotent stem cell line and differentiated cells thereof. In some embodiments, the plant cell includes a monocot cell or a dicot cell. In some embodiments, the monocot cell or the dicot cell includes rice cell, maize cell, or soybean cell.

[0300] According to an embodiment of the present application, the use of the nuclease described in the present application, the guide RNA described in the present application, the nucleic acid described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, the recombinant vector described in the present application, or the recombinant host cell described in the present application for introducing a double-strand break into a targeting gene of a host cell can be provided.

[0301] According to an embodiment of the present application, the use of the nuclease described in the present application, the guide RNA described in the present application, the nucleic acid described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, the recombinant vector described in the present application, or the recombinant host cell described in the present application for deleting, replacing or inserting a targeting gene of a host cell can be provided.

[0302] The host cell can be any host cell in which nucleases can be used. In some embodiments, the host cell includes, but is not limited to, an animal cell, a plant cell, an algal cell, a fungal cell, a yeast cell, or a bacterial cell. In some embodiments, the animal cell includes a mammalian cell. In some embodiments, the mammalian cell includes a primary cell (e.g., a mesenchymal stem cell, an endothelial cell, an epithelial cell, a fibroblast, a keratinocyte, a melanocyte, a smooth muscle cell, and an immune cell), an immortalized cell line (e.g., HEK293, NIH-3T3, RAW-264.7, STO, VERO, CT26, hTERT immortalized human endothelial / epithelial / fibroblast / keratinocyte / ductal / cell lines), a cancer cell line (e.g., Hela, HepG2 / 3, HL-60, HT-1080, HT-29, A549, SW620, HCT-15, HCT116, MDA-MB-231, MCF7, SK-OV-3, PANC-1, AsPc-1, THP-1, Huh7, KG-1, RAJI, HB-CB, Jurkat, K562, CRL5826, CHO, MDCK, and Renca), an embryonic stem cell line (e.g., H1, H9, WIBR2, WIBR3, G-Olig2, ESF158, RW. 4, R1, and D3) and differentiated cells thereof, or an induced pluripotent stem cell line and differentiated cells thereof. In some embodiments, the plant cell includes a monocot cell or a dicot cell. In some embodiments, the monocot cell or the dicot cell includes rice cell, maize cell, or soybean cell.

[0303] According to an embodiment of the present application, the use of the nuclease described in the present application, the guide RNA described in the present application, the nucleic acid described in the present application, the nucleic acid construct described in the present application, the composition described in the present application, the recombinant vector described in the present application, or the recombinant host cell described in the present application for preparing a drug or a preparation for gene therapy, cell therapy, genome research, and stem cell induction and post-induction differentiation can be provided.

[0304] The above various embodiments and preferences for the present application can be combined with each other (as long as they are not inherently contradictory to each other) and are suitable for the use of the present application, and the various embodiments formed by such combinations are considered as a part of the present application.EXAMPLES

[0305] Exemplary embodiments of the present application are described below in conjunction with the accompanying drawings, where various details of the examples of the present application are included to facilitate understanding. It should be understood that they are considered to be exemplary only and not intended to limit the protection scope of the present application. The protection scope of the present application is only defined by the claims. Therefore, those of ordinary skill in the art should be aware that various changes and modifications can be made to the examples described herein, without departing from the scope of the present application. Likewise, for clarity and conciseness, the description of well-known functions and structures is omitted in the following description.

[0306] Unless otherwise stated, the reagents and instruments used in the following examples are conventional products that are commercially available. Unless otherwise stated, experiments are performed under conventional conditions or conditions recommended by the manufacturer.Example 1: Construction of Nuclease Activity Detection System

[0307] A set of an RGS dual fluorescence surrogate reporter system was established to verify the activity of candidate nucleases.

[0308] Plasmid 1 consists of a complete set of elements capable of transcribing and expressing candidate nuclease proteins, comprising a constitutive promoter CMV (sequence as shown in SEQ ID NO: 405) that can initiate transcription in an eukaryotic cell, a candidate nuclease sequence (as shown in Table 1), a 5′-nuclear localization signal peptide sequence (sequence as shown in SEQ ID NO: 406), a 3′-nuclear localization signal peptide sequence (sequence as shown in SEQ ID NO: 407), a polyA sequence (sequence as shown in SEQ ID NO:408) that terminates transcription, and an ampicillin resistance gene sequence (sequence as shown in SEQ ID NO: 409).

[0309] Method for constructing plasmid 1: The amino acid sequence (or nucleotide sequence) of the candidate nuclease protein was synthesized through conventional gene synthesis by BGI Tech Solutions (Beijing Liuhe) Co., Ltd., with an ECORI cleavage site inserted into the upstream 5′ end of the sequence, and a BamH1 cleavage site inserted into the downstream 3′ end. Plasmid construction was also performed by the company responsible for the gene synthesis, and the specific construction method was as follows: 1. Preparation of vector. The plasmid backbone of a pcDNA3.1 plasmid vector was subjected to a double enzymatic cleavage digestion reaction using the single restriction endonuclease cleavage sites ECORI and BamHI on the plasmid vector, a linearized plasmid vector fragment was obtained by agarose gel electrophoresis, and the enzymatic cleavage band was excised from the gel for recovery to obtain the purified linearized plasmid vector fragment. 2. Ligation. The nucleotide sequence of the candidate nuclease protein obtained through conventional gene synthesis was ligated with the linearized pcDNA3.1 vector fragment using a T4 DNA ligase. 3. Transformation and verification. Monoclonal transformants were obtained through a LB agar plate for screening ampicillin resistance, and the correct clone identified by sequencing was used as a candidate plasmid for later use.

[0310] Plasmid 2 comprises a reRNA sequence (as shown in Table 1), with a 20 nt targeted sequence GCTCGGAGATCATCATTGCG inserted at the 3′ end of the reRNA sequence, a U6 promoter (sequence as shown in SEQ ID NO: 410), a PBR322 replication origin (sequence as shown in SEQ ID NO: 411), and an ampicillin resistance gene sequence (sequence as shown in SEQ ID NO: 409).

[0311] Method for constructing plasmid 2: Guide reRNA was synthesized through conventional gene synthesis by Beijing Tsingke Biotech Co., Ltd. or General Biosystems (Anhui) Co., Ltd. Plasmid construction was also performed by the company responsible for the gene synthesis, and the specific construction method was as follows: 1. Preparation of vector. A pUC19-U6 vector was subjected to enzymatic cleavage using BbsI, a linearized plasmid vector fragment was obtained by agarose gel electrophoresis, and the enzymatic cleavage band was excised from the gel for recovery to obtain the purified linearized plasmid vector fragment. 2. Ligation. The nucleotide sequence of the guide reRNA obtained through gene synthesis was ligated with the linearized pUC19-U6 vector fragment using a ligation method of seamless cloning. 3. Transformation and verification. Monoclonal transformants were obtained through a LB agar plate for screening ampicillin resistance, and the correct clone identified by sequencing was used as a candidate plasmid for later use.

[0312] Plasmid 3 comprises a TAM sequence (as shown in Table 1), with a 20 nt targeted sequence GCTCGGAGATCATCATTGCG inserted at the 3′ end of the TAM sequence, a CMV promoter (sequence as shown in SEQ ID NO: 412), an ampicillin resistance gene sequence (sequence as shown in SEQ ID NO: 409), and a surrogate reporter gene. The surrogate reporter gene can encode two fluorescent proteins (RFP sequence as shown in SEQ ID NO: 413, and GFP sequence as shown in SEQ ID NO: 414). By means of the insertion of an endonuclease downstream of RFP and the insertion of an endonuclease upstream of GFP, TAM and a 20 nt targeted sequence at the 3′ end of TAM can be recognized. When there is no endonuclease activity according to the detection system, the reporter gene only expresses RFP to indicate the reference gene expression level of the reporter system, while GFP is designed outside the open reading frame (ORF) and therefore is not expressed. When the candidate has endonuclease activity, it can induce a double-strand break at the targeting site before GFP, which leads to the frameshift mutation of the reading frame when DNA is repaired through non-homology end joining (NHEJ), resulting in GFP shifting from an out of frame state to an in frame state and beginning to express. The stronger the cleavage activity of a nuclease, the higher the proportion of GFP expressed after frameshift. Therefore, the expression intensity of GFP is positively correlated with the cleavage activity of the nuclease. The working mode of the detection system is as shown in FIG. 1.

[0313] Method for constructing plasmid 3: Through an oligo synthesis method, TAM, and a 20 nt targeted sequence with an ECORI enzymatic cleavage site inserted at the 5′ end of the upstream sequence and a BamH1 enzymatic cleavage site inserted at the 3′ end of the downstream sequence were subjected to whole synthesis. The specific construction was as follows: 1. Preparation of vector. The plasmid backbone of an RGS-pcDNA3.1 plasmid vector was subjected to a double enzymatic cleavage digestion reaction using the single restriction endonuclease cleavage sites ECORI and BamHI on the plasmid vector, a linearized plasmid vector fragment was obtained by agarose gel electrophoresis, and the enzymatic cleavage band was excised from the gel for recovery to obtain the purified linearized plasmid vector fragment. 2. Ligation. The nucleotide sequence of the guide reRNA obtained through gene synthesis was ligated with the linearized pUC19-U6 vector fragment using a ligation method of seamless cloning. 3. Transformation and verification. Monoclonal transformants were obtained through a LB agar plate for screening ampicillin resistance, and the correct clone identified by sequencing was used as a candidate plasmid for later use.TABLE 1Plasmid construction related sequencesNameNuclease sequencereRNA sequenceTAM sequenceTP_A_1SEQ ID NO: 1SEQ ID NO: 198TTATTP_A_2SEQ ID NO: 2SEQ ID NO: 199GCTACTP_A_8SEQ ID NO: 3SEQ ID NO: 200TTATTP_A_12SEQ ID NO: 4SEQ ID NO: 201TTATTP_A_18SEQ ID NO: 5SEQ ID NO: 202TTGATTP_B_18SEQ ID NO: 6SEQ ID NO: 203TTATTP_B_41SEQ ID NO: 7SEQ ID NO: 204TTATTP_B_46SEQ ID NO: 8SEQ ID NO: 205TTTATTP_B_70SEQ ID NO: 9SEQ ID NO: 206TGACTP_B_71SEQ ID NO: 10SEQ ID NO: 207TGATTP_B_72SEQ ID NO: 11SEQ ID NO: 208TGACTP_B_73SEQ ID NO: 12SEQ ID NO: 209TGATTP_C_23SEQ ID NO: 13SEQ ID NO: 210CCATTP_C_67SEQ ID NO: 14SEQ ID NO: 211TTTAATP_C_70SEQ ID NO: 15SEQ ID NO: 212TTACTP_C_74SEQ ID NO: 16SEQ ID NO: 213TTACTP_D_1SEQ ID NO: 17SEQ ID NO: 214TGACTP_D_3SEQ ID NO: 18SEQ ID NO: 215TGACTP_D_4SEQ ID NO: 19SEQ ID NO: 216TGACTP_D_8SEQ ID NO: 20SEQ ID NO: 217GCTACTP_D_17SEQ ID NO: 21SEQ ID NO: 218TTATTP_D_18SEQ ID NO: 22SEQ ID NO: 219TTATTP_D_23SEQ ID NO: 23SEQ ID NO: 220TTATTP_D_24SEQ ID NO: 24SEQ ID NO: 221TTACTP_D_25SEQ ID NO: 25SEQ ID NO: 222TTACTP_D_27SEQ ID NO: 26SEQ ID NO: 223TGATTP_D_30SEQ ID NO: 27SEQ ID NO: 224TTTAATP_D_32SEQ ID NO: 28SEQ ID NO: 225TTTAATP_D_40SEQ ID NO: 29SEQ ID NO: 226TGACTP_D_43SEQ ID NO: 30SEQ ID NO: 227TGATTP_D_51SEQ ID NO: 31SEQ ID NO: 228GCTACTP_D_59SEQ ID NO: 32SEQ ID NO: 229TTACTP_D_61SEQ ID NO: 33SEQ ID NO: 230TTACTP_D_66SEQ ID NO: 34SEQ ID NO: 231TTACTP_D_67SEQ ID NO: 35SEQ ID NO: 232TTACTP_D_71SEQ ID NO: 36SEQ ID NO: 233TTTAATP_D_72SEQ ID NO: 37SEQ ID NO: 234TTTAATP_D_73SEQ ID NO: 38SEQ ID NO: 235TTTAATP_E_2SEQ ID NO: 39SEQ ID NO: 236TTAGTP_E_15SEQ ID NO: 40SEQ ID NO: 237TTCAATP_E_17SEQ ID NO: 41SEQ ID NO: 238TCAATP_E_48SEQ ID NO: 42SEQ ID NO: 239TGATTP_F_56SEQ ID NO: 43SEQ ID NO: 240CCATTP_F_71SEQ ID NO: 44SEQ ID NO: 241GTGACTP_F_77SEQ ID NO: 45SEQ ID NO: 242TTTAATP_F_80SEQ ID NO: 46SEQ ID NO: 243TTTAATP_F_83SEQ ID NO: 47SEQ ID NO: 244TTTAATP_F_85SEQ ID NO: 48SEQ ID NO: 245TTTAATP_G_14SEQ ID NO: 49SEQ ID NO: 246TTAGTP_G_19SEQ ID NO: 50SEQ ID NO: 247TTATTP_G_20SEQ ID NO: 51SEQ ID NO: 248TTTATTP_G_24SEQ ID NO: 52SEQ ID NO: 249TTATTP_G_43SEQ ID NO: 53SEQ ID NO: 250TCACTP_G_52SEQ ID NO: 54SEQ ID NO: 251TCATTP_G_53SEQ ID NO: 55SEQ ID NO: 252TCATTP_G_61SEQ ID NO: 56SEQ ID NO: 253TTCATTP_G_66SEQ ID NO: 57SEQ ID NO: 254TTGAATP_G_72SEQ ID NO: 58SEQ ID NO: 255TGATTP_G_75SEQ ID NO: 59SEQ ID NO: 256TTTAATP_G_83SEQ ID NO: 60SEQ ID NO: 257TTATTP_G_84SEQ ID NO: 61SEQ ID NO: 258TTATTP_H_1SEQ ID NO: 62SEQ ID NO: 259TTTAATP_H_3SEQ ID NO: 63SEQ ID NO: 260TTTAATP_H_4SEQ ID NO: 64SEQ ID NO: 261TTTAATP_H_5SEQ ID NO: 65SEQ ID NO: 262TTTAATP_H_6SEQ ID NO: 66SEQ ID NO: 263TTTAATP_H_9SEQ ID NO: 67SEQ ID NO: 264TTTAATP_H_11SEQ ID NO: 68SEQ ID NO: 265TTTAATP_H_12SEQ ID NO: 69SEQ ID NO: 266TTTAATP_H_13SEQ ID NO: 70SEQ ID NO: 267TTTAATP_H_15SEQ ID NO: 71SEQ ID NO: 268TTTAATP_H_18SEQ ID NO: 72SEQ ID NO: 269TTTAATP_H_19SEQ ID NO: 73SEQ ID NO: 270TTTAATP_H_20SEQ ID NO: 74SEQ ID NO: 271TTTAATP_H_21SEQ ID NO: 75SEQ ID NO: 272TTTAATP_H_23SEQ ID NO: 76SEQ ID NO: 273TTTAATP_H_24SEQ ID NO: 77SEQ ID NO: 274TTTAATP_H_30SEQ ID NO: 78SEQ ID NO: 275TTTAATP_H_31SEQ ID NO: 79SEQ ID NO: 276TTTAATP_H_32SEQ ID NO: 80SEQ ID NO: 277TTTAATP_H_34SEQ ID NO: 81SEQ ID NO: 278TTTAATP_H_38SEQ ID NO: 82SEQ ID NO: 279TTTAATP_H_39SEQ ID NO: 83SEQ ID NO: 280TTTAATP_H_40SEQ ID NO: 84SEQ ID NO: 281TTTAATP_H_43SEQ ID NO: 85SEQ ID NO: 282TTTAATP_I_1SEQ ID NO: 86SEQ ID NO: 283CCATTP_I_2SEQ ID NO: 87SEQ ID NO: 284CCATTP_I_3SEQ ID NO: 88SEQ ID NO: 285CCATTP_1_4SEQ ID NO: 89SEQ ID NO: 286CCATTP_I_5SEQ ID NO: 90SEQ ID NO: 287CCATTP_1_6SEQ ID NO: 91SEQ ID NO: 288CCATTP_I_7SEQ ID NO: 92SEQ ID NO: 289CCATTP_I_8SEQ ID NO: 93SEQ ID NO: 290CCATTP_I_9SEQ ID NO: 94SEQ ID NO: 291CCATTP_I_10SEQ ID NO: 95SEQ ID NO: 292CCATTP_I_11SEQ ID NO: 96SEQ ID NO: 293CCATTP_I_12SEQ ID NO: 97SEQ ID NO: 294CCATTP_I_13SEQ ID NO: 98SEQ ID NO: 295CCATTP_1_15SEQ ID NO: 99SEQ ID NO: 296CCATTP_I_16SEQ ID NO: 100SEQ ID NO: 297CCATTP_I_17SEQ ID NO: 101SEQ ID NO: 298CCATTP_I_18SEQ ID NO: 102SEQ ID NO: 299CCATTP_I_19SEQ TD NO: 103SEQ ID NO: 300CCATTP_I_20SEQ ID NO: 104SEQ ID NO: 301CCATTP_I_21SEQ ID NO: 105SEQ ID NO: 302CCATTP_I_22SEQ ID NO: 106SEQ ID NO: 303CCATTP_I_24SEQ ID NO: 107SEQ ID NO: 304CCATTP_I_25SEQ ID NO: 108SEQ ID NO: 305CCATTP_I_26SEQ ID NO: 109SEQ ID NO: 306CCATTP_I_29SEQ ID NO: 110SEQ ID NO: 307CCATTP_I_31SEQ ID NO: 111SEQ ID NO: 308CCATTP_I_35SEQ ID NO: 112SEQ ID NO: 309CCATTP_I_37SEQ ID NO: 113SEQ ID NO: 310TTATTP_I_38SEQ ID NO: 114SEQ ID NO: 311TTATTP_I_40SEQ ID NO: 115SEQ ID NO: 312TTATTP_I_41SEQ ID NO: 116SEQ ID NO: 313TTATTP_I_44SEQ ID NO: 117SEQ ID NO: 314TTATTP_I_45SEQ ID NO: 118SEQ ID NO: 315TTATTP_I_46SEQ ID NO: 119SEQ ID NO: 316TTATTP_I_47SEQ ID NO: 120SEQ ID NO: 317TTATTP_I_48SEQ ID NO: 121SEQ ID NO: 318TTATTP_I_49SEQ ID NO: 122SEQ ID NO: 319TTATTP_I_50SEQ ID NO: 123SEQ ID NO: 320TTATTP_I_51SEQ ID NO: 124SEQ ID NO: 321TTATTP_I_52SEQ ID NO: 125SEQ ID NO: 322TTATTP_I_53SEQ ID NO: 126SEQ ID NO: 323TTATTP_I_55SEQ ID NO: 127SEQ ID NO: 324TTATTP_I_56SEQ ID NO: 128SEQ ID NO: 325TTATTP_I_58SEQ ID NO: 129SEQ ID NO: 326TTATTP_I_59SEQ ID NO: 130SEQ ID NO: 327TTATTP_I_61SEQ ID NO: 131SEQ ID NO: 328TTATTP_I_62SEQ ID NO: 132SEQ ID NO: 329TTATTP_I_64SEQ ID NO: 133SEQ ID NO: 330TTATTP_I_65SEQ ID NO: 134SEQ ID NO: 331TTATTP_I_66SEQ ID NO: 135SEQ ID NO: 332TTATTP_I_67SEQ ID NO: 136SEQ ID NO: 333TTATTP_I_70SEQ ID NO: 137SEQ ID NO: 334TTATTP_I_71SEQ ID NO: 138SEQ ID NO: 335TTATTP_I_76SEQ ID NO: 139SEQ ID NO: 336TTATTP_I_77SEQ TD NO: 140SEQ ID NO: 337TTATTP_I_80SEQ ID NO: 141SEQ ID NO: 338TTATTP_I_82SEQ ID NO: 142SEQ ID NO: 339TTATTP_I_84SEQ ID NO: 143SEQ ID NO: 340TTATTP_I_86SEQ ID NO: 144SEQ ID NO: 341TTATTP_I_87SEQ ID NO: 145SEQ ID NO: 342TTATTP_I_79SEQ ID NO: 146SEQ ID NO: 343TTATTP_I_85SEQ ID NO: 147SEQ ID NO: 344TTATTP_L_1SEQ ID NO: 148SEQ ID NO: 345TTTAATP_L_4SEQ ID NO: 149SEQ ID NO: 346TTTAATP_L_5SEQ TD NO: 150SEQ ID NO: 347TTTAATP_L_8SEQ ID NO: 151SEQ ID NO: 348TTTAATP_L_9SEQ ID NO: 152SEQ ID NO: 349TTTAATP_L_10SEQ ID NO: 153SEQ ID NO: 350TTTAATP_L_11SEQ ID NO: 154SEQ ID NO: 351TTTAATP_L_12SEQ ID NO: 155SEQ ID NO: 352TTTAATP_L_15SEQ ID NO: 156SEQ ID NO: 353TTTAATP_L_16SEQ ID NO: 157SEQ ID NO: 354TTTAATP_L_17SEQ ID NO: 158SEQ ID NO: 355TTTAATP_L_21SEQ ID NO: 159SEQ ID NO: 356TTTAATP_L_22SEQ ID NO: 160SEQ ID NO: 357TTTAATP_L_24SEQ ID NO: 161SEQ ID NO: 358TTTAATP_L_25SEQ ID NO: 162SEQ ID NO: 359TTTAATP_L_26SEQ ID NO: 163SEQ ID NO: 360TTTAATP_L_27SEQ ID NO: 164SEQ ID NO: 361TTTAATP_L_28SEQ ID NO: 165SEQ ID NO: 362TTTAATP_L_31SEQ ID NO: 166SEQ ID NO: 363TTTAATP_L_32SEQ ID NO: 167SEQ ID NO: 364TTTAATP_L_34SEQ ID NO: 168SEQ ID NO: 365TTTAATP_L_36SEQ ID NO: 169SEQ ID NO: 366TTTAATP_L_37SEQ ID NO: 170SEQ ID NO: 367TTTAATP_L_39SEQ ID NO: 171SEQ ID NO: 368TTTAATP_M_1SEQ ID NO: 172SEQ ID NO: 369TTGATTP_M_3SEQ ID NO: 173SEQ ID NO: 370TTGATTP_M_7SEQ ID NO: 174SEQ ID NO: 371TGACTP_M_11SEQ ID NO: 175SEQ ID NO: 372TGACTP_M_14SEQ ID NO: 176SEQ ID NO: 373TGACTP_M_17SEQ TD NO: 177SEQ ID NO: 374TGACTP_M_19SEQ ID NO: 178SEQ ID NO: 375TGACTP_M_20SEQ ID NO: 179SEQ ID NO: 376TGACTP_M_24SEQ TD NO: 180SEQ ID NO: 377TGACTP_M_31SEQ ID NO: 181SEQ TD NO: 378TTACTP_M_32SEQ ID NO: 182SEQ ID NO: 379TTACTP_M_33SEQ ID NO: 183SEQ LD NO: 380TTACTP_M_34SEQ ID NO: 184SEQ ID NO: 381TTACTP_M_35SEQ ID NO: 185SEQ ID NO: 382TTACTP_M_37SEQ ID NO: 186SEQ ID NO: 383TTAGTP_M_40SEQ ID NO: 187SEQ ID NO: 384TTACTP_M_41SEQ ID NO: 188SEQ ID NO: 385TTACTP_M_43SEQ ID NO: 189SEQ ID NO: 386TTACTP_M_46SEQ ID NO: 190SEQ ID NO: 387TCACTP_M_49SEQ ID NO: 191SEQ ID NO: 388TCACTP_M_58SEQ ID NO: 192SEQ ID NO: 389TCACTP_M_65SEQ ID NO: 193SEQ ID NO: 390TCACTP_M_66SEQ ID NO: 194SEQ ID NO: 391TCACTP_M_67SEQ ID NO: 195SEQ ID NO: 392TCACTP_M_70SEQ ID NO: 196SEQ ID NO: 393TCACTP_M_78SEQ ID NO: 197SEQ ID NO: 394TCACTP_A_24SEQ ID NO: 395SEQ ID NO: 400TTTAATP_A_54SEQ ID NO: 396SEQ ID NO: 401TTTAGTP_B_23SEQ ID NO: 397SEQ ID NO: 402TTGATTP_D_44SEQ ID NO: 398SEQ ID NO: 403TTGATTP_F_76SEQ ID NO: 399SEQ ID NO: 404ITTAAExample 2: Detection of Nuclease Activity2.1 Cell Treatment:

[0314] After HEK293T cells (commercially purchased) were cultured to the logarithmic growth phase, they were trypsinized into single cells with 0.25% Trypsin (Thermo), and added to a 96-well cell culture plate pre-coated with PDL (Sigma) at a cell concentration of 3× 104 cells / well, and cultured overnight at 37° C. in 5% CO2.2.2 Cell Transfection:

[0315] The three functional plasmids described in example 1 (the nuclease plasmid, the reRNA-targeted sequence plasmid and the RGS dual fluorescence reporter system plasmid) were co-transfected into HEK293T cells, wherein 60 ng of the nuclease plasmid, 40 ng of the reRNA-targeted sequence plasmid and 100 ng of the RGS dual fluorescence reporter system plasmid were added to a 96-well cell culture plate, respectively, and transfection was performed using Lipofectamine™ 2000 (Invitrogen, Cat. No. 11668019) at a ratio of transfection reagent volume (μL):plasmid mass (μg) of 2:1.2.3 Obtaining Results

[0316] After transfection, the cells were cultured for 48 h, then typsinized and collected, and detected by a flow cytometry. The final screening results were analyzed on the basis of the positive expression of GFP.2.4 Detection Results

[0317] The results of nuclease activity were obtained by flow cytometry, and the data were as shown in FIG. 2, with the ordinate in the figure showing the RFP expression (%) and the abscissa showing the GFP expression (%), which reflected the cleavage activity of the nuclease. Furthermore, the statistical results of the GFP expressions reflecting the activities of all nucleases were as shown in FIG. 3 and Table 2. The results showed that the 197 nucleases (TP_A_1, TP_A_2, TP_A_8, TP_A_12, TP_A_18, TP_B_18, TP_B_41, TP_B_46, TP_B_70, TP_B_71, TP_B_72, TP_B_73, TP_C_23, TP_C_67, TP_C_70, TP_C_74, TP_D_1, TP_D_3, TP_D_4, TP_D_8, TP_D_17, TP_D_18, TP_D_23, TP_D_24, TP_D_25, TP_D_27, TP_D_30, TP_D_32, TP_D_40, TP_D_43, TP_D_51, TP_D_59, TP_D_61, TP_D_66, TP_D_67, TP_D_71, TP_D_72, TP_D_73, TP_E_2, TP_E_15, TP_E_17, TP_E_48, TP_F_56, TP_F_71, TP_F_77, TP_F_80, TP_F_83, TP_F_85, TP_G_14, TP_G_19, TP_G_20, TP_G_24, TP_G_43, TP_G_52, TP_G_53, TP_G_61, TP_G_66, TP_G_72, TP_G_75, TP_G_83, TP_G_84, TP_H_1, TP_H_3, TP_H_4, TP_H_5, TP_H_6, TP_H_9, TP_H_11, TP_H_12, TP_H_13, TP_H_15, TP_H_18, TP_H_19, TP_H_20, TP_H_21, TP_H_23, TP_H_24, TP_H_30, TP_H_31, TP_H_32, TP_H_34, TP_H_38, TP_H_39, TP_H_40, TP_H_43, TP_I_1, TP_I_2, TP_I_3, TP_I_4, TP_I_5, TP_I_6, TP_I_7, TP_I_8, TP_I_9, TP_I_10, TP_I_11, TP_I_12, TP_I_13, TP_I_15, TP_I_16, TP_I_17, TP_I_18, TP_I_19, TP_I_20, TP_I_21, TP_I_22, TP_I_24, TP_I_25, TP_I_26, TP_I_29, TP_I_31, TP_I_35, TP_I_37, TP_I_38, TP_I_40, TP_I_41, TP_I_44, TP_I_45, TP_I_46, TP_I_47, TP_I_48, TP_I_49, TP_I_50, TP_I_51, TP_I_52, TP_I_53, TP_I_55, TP_I_56, TP_I_58, TP_I_59, TP_I_61, TP_I_62, TP_I_64, TP_I_65, TP_I_66, TP_I_67, TP_I_70, TP_I_71, TP_I_76, TP_I_77, TP_I_79, TP_I_80, TP_I_82, TP_I_84, TP_I_85, TP_I_86, TP_I_87, TP_L_1, TP_L_4, TP_L_5, TP_L_8, TP_L_9, TP_L_10, TP_L_11, TP_L_12, TP_L_15, TP_L_16, TP_L_17, TP_L_21, TP_L_22, TP_L_24, TP_L_25, TP_L_26, TP_L_27, TP_L_28, TP_L_31, TP_L_32, TP_L_34, TP_L_36, TP_L_37, TP_L_39, TP_M_1, TP_M_3, TP_M_7, TP_M_11, TP_M_14, TP_M_17, TP_M_19, TP_M_20, TP_M_24, TP_M_31, TP_M_32, TP_M_33, TP_M_34, TP_M_35, TP_M_37, TP_M_40, TP_M_41, TP_M_43, TP_M_46, TP_M_49, TP_M_58, TP_M_65, TP_M_66, TP_M_67, TP_M_70 and TP_M_78) in the present application had good activity.

[0318] Meanwhile, a large number of nucleases with inactive or low cleavage activity were also found during the screening process (e.g. TP_A_24, TP_A_54, TP_B_23, TP_D_44, and TP_F_76 in Table 1 of this application). Compared with these nucleases with inactive or low activity, the cleavage activity of the 197 nucleases of the present application were markedly higher.

[0319] In addition, FIG. 11 showed an evolutionary branching diagram of the nucleases in the present application based on protein sequences. The result showed that these nucleases covered different branches of the superfamily, and ISDra2 was also included.TABLE 2The results of nuclease activity in example 2Nuclease activityName(mean ± standard deviation)TP_A_13.92 ± 0.44TP_A_29.70 ± 1.46TP_A_82.78 ± 0.15TP_A_126.10 ± 0.30TP_A_182.36 ± 0.40TP_B_184.10 ± 0.78TP_B_410.66 ± 0.16TP_B_461.18 ± 0.13TP_B_700.95 ± 0.13TP_B_7119.94 ± 0.06 TP_B_722.66 ± 1.31TP_B_7322.61 ± 0.01 TP_C_2323.83 ± 1.97 TP_C_678.39 ± 2.07TP_C_7011.56 ± 1.23 TP_C_743.16 ± 0.12TP_D_129.99 ± 1.48 TP_D_325.85 ± 0.28 TP_D_44.44 ± 0  TP_D_85.53 ± 0.12TP_D_176.29 ± 0  TP_D_1811.25 ± 0.71 TP_D_234.66 ± 1.51TP_D_2411.89 ± 0.71 TP_D_256.61 ± 0  TP_D_2718.14 ± 0.78 TP_D_306.20 ± 1.93TP_D_329.84 ± 2.88TP_D_4022.01 ± 1.27 TP_D_4317.44 ± 4.17 TP_D_5129.25 ± 1.34 TP_D_592.81 ± 0.21TP_D_6115.55 ± 0.14 TP_D_664.27 ± 0  TP_D_6717.73 ± 1.48 TP_D_7114.06 ± 0.64 TP_D_7215.62 ± 6.36 TP_D_7313.55 ± 0.78 TP_E_27.03 ± 1.84TP_E_1513.41 ± 6.99 TP_E_1713.41 ± 5.44 TP_E_4810.37 ± 0   TP_F_5613.65 ± 1.09 TP_F_719.91 ± 0.27TP_F_771.91 ± 0.04TP_F_803.59 ± 1.51TP_F_831.38 ± 0.09TP_F_859.67 ± 0.16TP_G_1420.62 ± 0.34 TP_G_1915.99 ± 0.87 TP_G_204.64 ± 0.12TP_G_2420.69 ± 1.04 TP_G_4315.33 ± 1.07 TP_G_526.75 ± 0.09TP_G_534.42 ± 0.11TP_G_6110.36 ± 0.33 TP_G_662.41 ± 0.21TP_G_7218.87 ± 0   TP_G_755.53 ± 0.17TP_G_836.50 ± 1.00TP_G_843.35 ± 0.07TP_H_18.11 ± 0.94TP_H_311.32 ± 8.28 TP_H_43.01 ± 0.08TP_H_512.54 ± 5.42 TP_H_610.25 ± 2.01 TP_H_916.47 ± 5.23 TP_H_1115.42 ± 0.07 TP_H_125.88 ± 0.10TP_H_135.74 ± 1.20TP_H_156.02 ± 1.40TP_H_185.88 ± 0.94TP_H_197.93 ± 1.90TP_H_206.17 ± 2.12TP_H_215.12 ± 0.15TP_H_235.29 ± 1.75TP_H_2411.74 ± 3.59 TP_H_3012.57 ± 1.98 TP_H_316.94 ± 0.43TP_H_3214.02 ± 4.45 TP_H_3415.37 ± 6.36 TP_H_3813.62 ± 3.75 TP_H_395.74 ± 1.44TP_H_407.45 ± 1.87TP_H_434.79 ± 0.23TP_I_123.47 ± 2.46 TP_I_220.17 ± 2.18 TP_I_36.26 ± 0.93TP_I_47.27 ± 0.38TP_I_519.67 ± 2.60 TP_I_622.97 ± 0.37 TP_I_75.72 ± 0.45TP_I_810.32 ± 0.13 TP_I_927.07 ± 1.50 TP_I_1011.47 ± 1.36 TP_I_1120.92 ± 0.86 TP_I_1226.02 ± 0.41 TP_I_134.65 ± 0.09TP_I_1531.77 ± 1.61 TP_I_167.86 ± 0.20TP_I_1717.47 ± 0.79 TP_I_1827.32 ± 0.98 TP_I_194.95 ± 0.21TP_I_2022.72 ± 1.29 TP_I_2121.52 ± 0.58 TP_I_2211.62 ± 0.27 TP_I_249.92 ± 0.27TP_I_258.37 ± 0.58TP_I_264.58 ± 0.49TP_I_299.77 ± 0.06TP_I_3110.32 ± 0.27 TP_I_3512.32 ± 0.01 TP_I_3715.32 ± 0.16 TP_I_3817.57 ± 1.61 TP_I_4012.12 ± 0.58 TP_I_4117.62 ± 0.44 TP_I_4415.32 ± 1.15 TP_I_4517.37 ± 0.34 TP_I_4612.97 ± 0.51 TP_I_4716.32 ± 0.16 TP_I_485.77 ± 0.74TP_I_4918.27 ± 1.78 TP_I_506.47 ± 0.47TP_I_5115.67 ± 1.05 TP_I_523.55 ± 0.18TP_I_534.40 ± 0.42TP_I_555.21 ± 0.71TP_I_561.96 ± 0.16TP_I_583.85 ± 0.48TP_I_598.22 ± 0.65TP_I_617.36 ± 1.98TP_I_623.69 ± 0.47TP_I_6423.57 ± 1.22 TP_I_656.70 ± 0.48TP_I_663.18 ± 0.17TP_I_672.60 ± 0.01TP_I_7010.21 ± 1.45 TP_I_713.84 ± 0.06TP_I_7612.62 ± 1.57 TP_I_7711.97 ± 1.19 TP_I_8019.52 ± 1.15 TP_I_823.80 ± 1.04TP_I_845.49 ± 0.06TP_I_8612.97 ± 1.07 TP_I_879.93 ± 1.39TP_I_793.64 ± 1.52TP_I_853.64 ± 0.40TP_L_13.24 ± 0.79TP_L_424.19 ± 3.80 TP_L_518.79 ± 0.27 TP_L_814.69 ± 1.82 TP_L_913.44 ± 2.63 TP_L_105.07 ± 0.25TP_L_1113.14 ± 1.36 TP_L_1213.24 ± 0.91 TP_L_1514.94 ± 0.62 TP_L_164.67 ± 0.33TP_L_1716.19 ± 2.56 TP_L_2116.64 ± 2.91 TP_L_2221.84 ± 0.93 TP_L_2410.79 ± 0.01 TP_L_257.68 ± 0.10TP_L_263.24 ± 0.31TP_L_2719.54 ± 0.48 TP_L_284.83 ± 0.45TP_L_318.91 ± 1.12TP_L_3213.59 ± 0.16 TP_L_344.09 ± 0.78TP_L_3613.14 ± 0.93 TP_L_378.00 ± 1.20TP_L_3921.79 ± 0.72 TP_M_12.59 ± 0.30TP_M_323.78 ± 1.16 TP_M_721.65 ± 0.92 TP_M_112.52 ± 0.30TP_M_1419.35 ± 2.62 TP_M_175.69 ± 0.30TP_M_19 1.66 ± 10.11TP_M_208.51 ± 0.96TP_M_241.90 ± 0.08TP_M_3117.35 ± 3.49 TP_M_3219.40 ± 0.73 TP_M_336.75 ± 1.16TP_M_3417.50 ± 2.43 TP_M_3510.15 ± 0.37 TP_M_372.15 ± 0.18TP_M_406.67 ± 1.19TP_M_412.26 ± 0.21TP_M_434.97 ± 0.24TP_M_467.25 ± 0.61TP_M_491.22 ± 0.21TP_M_5814.35 ± 0.21 TP_M_654.35 ± 0.40TP_M_663.41 ± 1.68TP_M_6711.16 ± 0.47 TP_M_701.03 ± 0.01TP_M_781.94 ± 0.50TP_A_240.25 ± 0.08TP_A_540.11 ± 0.09TP_B_230.24 ± 0.08TP_D_440.18 ± 0  TP_F_760.48 ± 0.34Example 3: Detection of Editing Efficiency at Endogenous Loci3.1 Construction of Plasmids:

[0320] The nuclease plasmid (plasmid 1) comprised a complete set of elements capable of transcribing and expressing candidate nuclease proteins, including a constitutive promoter CMV (sequence as shown in SEQ ID NO: 405) that can initiate transcription in an eukaryotic cell, a candidate nuclease sequence (as shown in Table 1), a 5′-nuclear localization signal peptide sequence (sequence as shown in SEQ ID NO: 406), a 3′-nuclear localization signal peptide sequence (sequence as shown in SEQ ID NO: 407), a polyA sequence (sequence as shown in SEQ ID NO: 408) that terminates transcription, and an ampicillin resistance gene sequence (sequence as shown in SEQ ID NO: 409). The method for constructing plasmid 1 is described in example 1.

[0321] The reRNA-targeted sequence plasmid (plasmid 4) comprises a reRNA sequence (as shown in Table 1), with a 20 nt targeted sequence of endogenous gene inserted at the 3′ end of the reRNA sequence (as shown in Table 3), a U6 promoter (sequence as shown in SEQ ID NO: 410), a PBR322 replication origin (sequence as shown in SEQ ID NO: 411), and an ampicillin resistance gene sequence (sequence as shown in SEQ ID NO: 409). In addition, different targeted sequences of endogenous genes (as shown in Table 2) can identify different targeting genes adjacent to the TAM sequences.Method for Constructing Plasmid 4:1. Preparation of vector. A reRNA plasmid containing BBSI-BBSI fragment (pUC19-U6-reRNA-BbsI_BbsI) was subjected to enzymatic cleavage using BbsI, a linearized plasmid vector fragment was obtained by agarose gel electrophoresis, and the enzymatic cleavage band was excised from the gel for recovery to obtain the purified linearized plasmid vector fragment. 2. Preparation of 20 nt targeted sequences of endogenous genes. Firstly, the 20 bp DNA sequence adjacent to the 3′ end of the TAM sequence was searched in the endogenous gene sequence, and then oligonucleotides of targeted sequences with BbsI excision end were synthesized through primer synthesis. Finally, a double-stranded oligonucleotide with sticky ends was synthesized by annealing bonding. 3. Ligation. The targeted sequence of endogenous gene was ligated with the linearized pUC19-U6-reRNA-BbsI_BbsI vector fragment using T4 ligase. 4. Transformation and verification. Monoclonal transformants were obtained through a LB agar plate for screening ampicillin resistance, and the correct clone identified by sequencing was used as a candidate plasmid for later use.3.2 Cell Treatment:

[0323] After HEK293T cells (commercially purchased) were cultured to the logarithmic growth phase, they were typsinized into single cells with 0.25% Trypsin (Thermo), and added to a 48-well cell culture plate pre-coated with PDL (Sigma) at a cell concentration of 1× 105 cells / well, and cultured overnight at 37° C. in 5% CO2.3.3 Cell Transfection:

[0324] The two functional plasmids described in 3.1 (the nuclease plasmid and the reRNA-targeted sequence plasmid) were co-transfected into HEK293T cells, wherein 300 ng of the nuclease plasmid and 200 ng of the reRNA-targeted sequence plasmid were added to a 48-well cell culture plate, respectively, and transfection was performed using Lipofectamine™ 2000 (Invitrogen, Cat. No. 11668019) at a ratio of transfection reagent volume (μL):plasmid mass (μg) of 2:1.3.4 PCR Amplification and NGS Second Generation Sequencing

[0325] After transfection, the cells were cultured for 48 h, then typsinized and collected, and the genome DNA was extracted. PCR primers were designed near the targeted sequence of endogenous gene to amplify a length of about 200 bp PCR product including 20 nt targeted sequence. The PCR products were sequenced by the next generation sequencing.3.5 Detection Results

[0326] The results of endogenous gene editing efficiency were as shown in FIG. 9 and Table 3.

[0327] By analyzing the sequence data generated by the next generation sequencing technology, the endogenous gene editing activity of nuclease was determined by counting the base insertions and deletions (Indel %) generated on the targeted sequence of endogenous gene. The results showed that the nucleases in this application showed good editing efficiency on different endogenous genes.

[0328] It should be stated that the above are only the preferred examples of the present application and are not intended to limit the present application. For those of ordinary skill in the art, various modifications and changes can be made to the present application. Although the specific embodiments have been described, for the applicant or a person skilled in the art, the substitutions, modifications, changes, improvements, and substantial equivalents of the above embodiments may exist or cannot be foreseen currently. Therefore, the submitted appended claims and claims that may be modified are intended to cover all such substitutions, modifications, changes, improvements, and substantial equivalents. It is important that, as the technology evolves, many elements described herein may be replaced with equivalent elements that appear after the present application.TABLE 3The results of endogenous gene editing activity in example 3Nuclease Targeted sequence ofTargeting Endogenous gene nameendogenous genegeneediting activityTP_C_23SEQ ID NO: 415CEP29021.860SEQ ID NO: 416HBG6.821SEQ ID NO: 417KLKB122.947SEQ ID NO: 418TET117.241SEQ ID NO: 419TTR5.520TP_D_51SEQ ID NO: 420PGK141.613SEQ ID NO: 421AAVS126.304SEQ ID NO: 422B2M14.865TP_D_67SEQ ID NO: 423AAVS119.088SEQ ID NO: 424B2M9.761SEQ ID NO: 425TET18.216SEQ ID NO: 426TTR18.903TP_E_15SEQ ID NO: 427TET118.951SEQ ID NO: 428TRAC8.351SEQ ID NO: 429DNMT16.336SEQ ID NO: 430EMX14.493TP_F_85SEQ ID NO: 431BCL  1A6.215SEQ ID NO: 432KLKB16.420TP_G_24SEQ ID NO: 433TET234.649SEQ ID NO: 434TET126.251SEQ ID NO: 435CEP29023.352SEQ ID NO: 436TTR19.559SEQ ID NO: 437BCL11A21.510SEQ ID NO: 438KLKB116.525SEQ ID NO: 439CD5214.159SEQ ID NO: 440AAVS111.543TP_H_5SEQ ID NO: 441PGK17.174SEQ ID NO: 442TET18.492TP_H_6SEQ ID NO: 443PGK24.552SEQ ID NO: 444BCL11A12.442SEQ ID NO: 445TET113.245SEQ ID NO: 446B2M8.950SEQ ID NO: 447TET26.501TP_H_9SEQ ID NO: 448BCL11A7.978SEQ ID NO: 449KLKB17.351SEQ ID NO: 450B2M5.903SEQ ID NO: 451PGK4.980SEQ ID NO: 452TET  4.783TP_H_11SEQ ID NO: 453B2M61.666SEQ ID NO: 454BCL11A54.506SEQ ID NO: 455KLKB162.239SEQ ID NO: 456PGK54.994SEQ ID NO: 457TET150.273SEQ ID NO: 458TET261.705SEQ ID NO: 459TRAC53.345SEQ ID NO: 460TTR50.796SEQ ID NO: 461CD713.834TP_H_24SEQ ID NO: 462PGK20.581SEQ ID NO: 463KLKB118.775SEQ ID NO: 464CD713.384SEQ ID NO: 465TET  10.356SEQ ID NO: 466TRAC9.612SEQ ID NO: 467B2M9.692SEQ ID NO: 468BCL11A5.489TP_H_30SEQ ID NO: 469PGK20.436SEQ ID NO: 470TET116.469SEQ ID NO: 471CD712.278SEQ ID NO: 472KLKB113.315SEQ ID NO: 473TRAC7.763SEQ ID NO: 474B2M4.450SEQ ID NO: 475TET25.619TP_H_32SEQ ID NO: 476KLKB151.101SEQ ID NO: 477PGK42.658SEQ ID NO: 478TET119.984SEQ ID NO: 479TET219.217SEQ ID NO: 480TRAC23.268SEQ ID NO: 481BCL11A17.306SEQ ID NO: 482TTR7.225SEQ ID NO: 483B2M6.167TP_H_34SEQ ID NO: 484KLKB130.825SEQ ID NO: 485PGK27.967SEQ ID NO: 486TET117.912SEQ ID NO: 487TRAC16.485SEQ ID NO: 488TTR14.895SEQ ID NO: 489B2M14.660SEQ ID NO: 490TET212.292SEQ ID NO: 491BCL11A6.957TP_H_38SEQ ID NO: 492KLKB163.297SEQ ID NO: 493PGK46.872SEQ ID NO: 494B2M45.881SEQ ID NO: 495TET232.409SEQ ID NO: 496TET130.409SEQ ID NO: 497TRAC29.131SEQ ID NO: 498TTR27.788SEQ ID NO: 499BCL11A25.266SEQ ID NO: 500CD718.525TP_I_1SEQ ID NO: 501TET159.225SEQ ID NO: 502CEP29057.060SEQ ID NO: 503KLKB155.270SEQ ID NO: 504AAVS138.955SEQ ID NO: 505B2M39.494SEQ ID NO: 506HBG1_235.612SEQ ID NO: 507TTR29.442SEQ ID NO: 508TET221.489SEQ ID NO: 509TRAC13.207TP_I_5SEQ ID NO: 510KLKB155.169SEQ ID NO: 511TET147.776SEQ ID NO: 512CEP29034.743SEQ ID NO: 513AAVS126.766SEQ ID NO: 514TTR25.127SEQ ID NO: 515HBG1_221.574SEQ ID NO: 516B2M19.316SEQ ID NO: 517TET212.666TP_I_6SEQ ID NO: 518TET150.081SEQ ID NO: 519HBG1_237.687SEQ ID NO: 520TET231.753SEQ ID NO: 521KLKB129.755SEQ ID NO: 522B2M23.804SEQ ID NO: 523TTR20.689SEQ ID NO: 524AAVS118.009SEQ ID NO: 525CEP29017.971TP_I_12SEQ ID NO: 526TET153.183SEQ ID NO: 527TET246.974SEQ ID NO: 528KLKB146.170SEQ ID NO: 529CEP29046.364SEQ ID NO: 530TRAC47.434SEQ ID NO: 531B2M34.654SEQ ID NO: 532HBG1_233.385SEQ ID NO: 533TTR28.052SEQ ID NO: 534AAVS122.908SEQ ID NO: 535PD_112.380TP_I_15SEQ ID NO: 536CEP29088.371SEQ ID NO: 537HBG1 / 255.103SEQ ID NO: 538TET264.135SEQ ID NO: 539KLKB148.094SEQ ID NO: 540B2M44.417SEQ ID NO: 541TET142.320SEQ ID NO: 542AAVS141.814SEQ ID NO: 543TRAC41.858SEQ ID NO: 544TTR37.542SEQ ID NO: 545PD  28.804TP_I_18SEQ ID NO: 546CEP29061.648SEQ ID NO: 547TET158.509SEQ ID NO: 548TET251.308SEQ ID NO: 549TTR49.338SEQ ID NO: 550TRAC43.432SEQ ID NO: 551AAVS140.697SEQ ID NO: 552B2M38.666SEQ ID NO: 553KLKB139.519SEQ ID NO: 554HBG1 / 233.877TP_I_20SEQ ID NO: 555TET163.758SEQ ID NO: 556CEP29063.903SEQ ID NO: 557KLKB161.443SEQ ID NO: 558B2M56.965SEQ ID NO: 559AAVS134.039SEQ ID NO: 560HBG1 / 239.727SEQ ID NO: 561TET239.042SEQ ID NO: 562TTR25.227SEQ ID NO: 563TRAC12.553SEQ ID NO: 564PD19.582TP_I_38SEQ ID NO: 565AAVS148.722SEQ ID NO: 566B2M45.242SEQ ID NO: 567BCL11A59.706SEQ ID NO: 568CD5253.727SEQ ID NO: 569KLKB137.065SEQ ID NO: 570TET252.323SEQ ID NO: 571TTR47.838TP_I_49SEQ ID NO: 572CD5254.098SEQ ID NO: 573AAVS151.949SEQ ID NO: 574TET249.506SEQ ID NO: 575BCL11A48.896SEQ ID NO: 576KLKB142.187SEQ ID NO: 577TTR42.214SEQ ID NO: 578B2M24.369TP_I_64SEQ ID NO: 579TET242.606SEQ ID NO: 580BCL11A31.702SEQ ID NO: 581AAVS119.840SEQ ID NO: 582KLKB119.125SEQ ID NO: 583CD5218.134SEQ ID NO: 584TTR18.923SEQ ID NO: 585B2M14.583TP_I_79SEQ ID NO: 586KLKB137.171SEQ ID NO: 587BCL11A36.493SEQ ID NO: 588TET234.402SEQ ID NO: 589CD5224.807SEQ ID NO: 590B2M24.635SEQ ID NO: 591TTR21.356SEQ ID NO: 592AAVS117.943 indicates data missing or illegible when filedExample 4: Detection of the Nuclease Activity in Rice Protoplasts

[0329] In this example, nuclease activity in rice protoplasts was evaluated using a pair of synthetic YFP gene report vectors (plasmids 5 and 6), which were constructed using the method described in example 1 (as shown in FIG. 13).

[0330] Plasmid 5 comprising a promoter ZmUBI (SEQ ID NO: 593), a candidate nuclease sequence (as shown in Table 1), a NOS terminator (SEQ ID NO: 594), a promoter OsU6 (SEQ ID NO: 595), a reRNA sequence corresponding to a specific nuclease (as shown in Table 1), a spacer sequence (SEQ ID NO: 596), and a terminator (SEQ ID NO: 597).

[0331] In plasmid 6, the YFP sequence (SEQ ID NO: 598) was segmented by spacer sequence (SEQ ID NO: 596) and the TAM sequence (as shown in Table 1) corresponding to a specific nuclease in plasmid 5. And The YFP sequence in the first half overlapped with the YFP sequence in the second half. Plasmid 6 also comprising a promoter 35S (SEQ ID NO: 599) and a terminator (SEQ ID NO: 600).

[0332] After co-transforming plasmid 5 and plasmid 6 into rice protoplasts, once the spacer sequence in plasmid 6 is cut by nuclease, the partially overlapping fragment (derived from the middle segment of YFP) promotes DSB repair through homologous dependent DNA repair pathway, thus restoring normal YFP gene (as shown in FIG. 14). Therefore, the cleavage activity of nuclease can be evaluated by observing the number of YFP-positive cells.

[0333] The results of YFP fluorescence were as shown in FIG. 15-31, which showed that the 197 nucleases in the present application had good cleavage activity in rice protoplasts as well (TP_A_1, TP_A_2, TP_A_8, TP_A_12, TP_A_18, TP_B_18, TP_B_41, TP_B_46, TP_B_70, TP_B_71, TP_B_72, TP_B_73, TP_C_23, TP_C_67, TP_C_70, TP_C_74, TP_D_1, TP_D_3, TP_D_4, TP_D_8, TP_D_17, TP_D_18, TP_D_23, TP_D_24, TP_D_25, TP_D_27, TP_D_30, TP_D_32, TP_D_40, TP_D_43, TP_D_51, TP_D_59, TP_D_61, TP_D_66, TP_D_67, TP_D_71, TP_D_72, TP_D_73, TP_E_2, TP_E_15, TP_E_17, TP_E_48, TP_F_56, TP_F_71, TP_F_77, TP_F_80, TP_F_83, TP_F_85, TP_G_14, TP_G_19, TP_G_20, TP_G_24, TP_G_43, TP_G_52, TP_G_53, TP_G_61, TP_G_66, TP_G_72, TP_G_75, TP_G_83, TP_G_84, TP_H_1, TP_H_3, TP_H_4, TP_H_5, TP_H_6, TP_H_9, TP_H_11, TP_H_12, TP_H_13, TP_H_15, TP_H_18, TP_H_19, TP_H_20, TP_H_21, TP_H_23, TP_H_24, TP_H_30, TP_H_31, TP_H_32, TP_H_34, TP_H_38, TP_H_39, TP_H_40, TP_H_43, TP_I_1, TP_I_2, TP_I_3, TP_I_4, TP_I_5, TP_I_6, TP_I_7, TP_I_8, TP_I_9, TP_I_10, TP_I_11, TP_I_12, TP_I_13, TP_I_15, TP_I_16, TP_I_17, TP_I_18, TP_I_19, TP_I_20, TP_I_21, TP_I_22, TP_I_24, TP_I_25, TP_I_26, TP_I_29, TP_I_31, TP_I_35, TP_I_37, TP_I_38, TP_I_40, TP_I_41, TP_I_44, TP_I_45, TP_I_46, TP_I_47, TP_I_48, TP_I_49, TP_I_50, TP_I_51, TP_I_52, TP_I_53, TP_I_55, TP_I_56, TP_I_58, TP_I_59, TP_I_61, TP_I_62, TP_I_64, TP_I_65, TP_I_66, TP_I_67, TP_I_70, TP_I_71, TP_I_76, TP_I_77, TP_I_79, TP_I_80, TP_I_82, TP_I_84, TP_I_85, TP_I_86, TP_I_87, TP_L_1, TP_L_4, TP_L_5, TP_L_8, TP_L_9, TP_L_10, TP_L_11, TP_L_12, TP_L_15, TP_L_16, TP_L_17, TP_L_21, TP_L_22, TP_L_24, TP_L_25, TP_L_26, TP_L_27, TP_L_28, TP_L_31, TP_L_32, TP_L_34, TP_L_36, TP_L_37, TP_L_39, TP_M_1, TP_M_3, TP_M_7, TP_M_11, TP_M_14, TP_M_17, TP_M_19, TP_M_20, TP_M_24, TP_M_31, TP_M_32, TP_M_33, TP_M_34, TP_M_35, TP_M_37, TP_M_40, TP_M_41, TP_M_43, TP_M_46, TP_M_49, TP_M_58, TP_M_65, TP_M_66, TP_M_67, TP_M_70 and TP_M_78).

Claims

1. An isolated nuclease, wherein the nuclease comprises an amino acid sequence as shown in the following formula:(X1)(X2)a(X3)(X4)(X5)b(X6)(X7)c(X8)(X9)d(X10)(X11)e(X12)(X13)f(X14)(X15)g(X16)wherein,a, b, c, d, e, f, and g are the numbers of amino acids;(X1), (X3), (X4), (X6), (X8), (X10), (X12), (X14), and (X16) are independently polar amino acids or aliphatic amino acids;(X2) is any amino acid, and a is 15 or 16;(X5) is any amino acid, and b is 2;(X7) is any amino acid, and c is 2, 3 or 4;(X9) is any amino acid, and dis 14, 15, 16, 17 or 18;(X11) is any amino acid, and e is 1 or 2;(X13) is any amino acid, and f is 6; and(X15) is any amino acid, and g is 5.

2. The nuclease according to claim 1, wherein the(X1) is a positively charged amino acid;(X3) is a polar uncharged amino acid;(X4) is a polar uncharged amino acid;(X6) is a polar uncharged amino acid;(X8) is a polar uncharged amino acid;(X10) is a polar uncharged amino acid;(X12) is a polar uncharged amino acid;(X14) is a negatively charged amino acid; and(X16) is a polar uncharged amino acid.

3. The nuclease according to claim 2, wherein the (X1) is K; (X3) is S or T; (X4) is S or T; (X6) is C; (X8) is C; (X10) is C; (X12) is C; (X14) is D; and (X16) is N.4.-11. (canceled)12. An isolated nuclease, wherein the nuclease has a nuclease sequence selected from the following (i) or a variant sequence of the aforementioned nuclease having a nuclease activity in (ii)-(iv):(i) at least one amino acid sequence as shown in any one of SEQ ID NOs: 1-197;(ii) at least one of sequences obtained by performing deletion, substitution, insertion, or mutation of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids on the amino acid sequence as shown in any one of SEQ ID NOs: 1-197;(iii) at least one of amino acid sequences having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence as shown in any one of SEQ ID NOs: 1-197; and(iv) at least one of sequences obtained by further fusing the amino acid sequence as shown in any one of SEQ ID NOs: 1-197 with other sequences.

13. The nuclease according to claim 12, wherein the nuclease has a nuclease sequence selected from at least one of the following groups (1)-(9):(1) at least one amino acid sequence as shown in any one of SEQ ID NOs: 52 and 113-147;(2) at least one amino acid sequence as shown in any one of SEQ ID NOs: 27-28, 36-38, 62-85 and 148-171;(3) at least one amino acid sequence as shown in any one of SEQ ID NOs: 13, 86-100 and 105-110;(4) at least one amino acid sequence as shown in any one of SEQ ID NOs: 10-11, 17-19, 29-30 and 174-180;(5) at least one amino acid sequence as shown in any one of SEQ ID NOs: 34, 35, 50, 61 and 181-189;(6) at least one amino acid sequence as shown in any one of SEQ ID NOs: 53 and 190-197;(7) at least one amino acid sequence as shown in any one of SEQ ID NOs: 101, 103, 104 and 112;(8) at least one amino acid sequence as shown in any one of SEQ ID NOs: 7 and 23-25; and(9) at least one amino acid sequence as shown in any one of SEQ ID NOs: 3, 21 and 22.

14. The nuclease according to claim 12, wherein the nuclease has a nuclease sequence selected from at least one of the following groups (1)-(12):(1) at least one amino acid sequence as shown in any one of SEQ ID NOs: 1, 3-4, 6-7, 21-23, 50, 52, 60-61 and 113-147;(2) at least one amino acid sequence as shown in any one of SEQ ID NOs: 14, 27-28, 36-38, 45-48, 59, 62-85 and 148-171;(3) at least one amino acid sequence as shown in any one of SEQ ID NOs: 13, 43 and 86-112;(4) at least one amino acid sequence as shown in any one of SEQ ID NOs: 15-16, 24-25, 32-35 and 181-189;(5) at least one amino acid sequence as shown in any one of SEQ ID NOs: 9, 11, 17-19, 29 and 174-180;(6) at least one amino acid sequence as shown in any one of SEQ ID NOs: 10, 12, 26, 30, 42 and 58;(7) at least one amino acid sequence as shown in any one of SEQ ID NOs: 2, 20 and 31;(8) at least one amino acid sequence as shown in any one of SEQ ID NOs: 8 and 51;(9) at least one amino acid sequence as shown in any one of SEQ ID NOs: 39 and 49;(10) at least one amino acid sequence as shown in any one of SEQ ID NOs: 54 and 55;(11) at least one amino acid sequence as shown in any one of SEQ ID NOs: 53 and 190-197; and(12) at least one amino acid sequence as shown in any one of SEQ ID NOs: 5, 172 and 173.15.-18. (canceled)19. A guide RNA, wherein the guide RNA comprises a reRNA, the reRNA comprises a nucleotide sequence as shown in any one of SEQ ID NOs: 198-394 or a variant thereof, and the guide RNA can bind to a specific nuclease.

20. The guide RNA according to claim 19, wherein the reRNA comprises at least one of nucleotide sequences having at least 70%, 80%, 90%, 95% or 99% identity to the nucleotide sequence as shown in any one of SEQ ID NOs: 198-394.21.-22. (canceled)23. The guide RNA according to claim 20, wherein the guide RNA further comprises a targeted sequence that can recognize a targeting gene adjacent to a transposon-associated motif.

24. The guide RNA according to claim 23, wherein the targeted sequence is of at least one of 10-50, 10-40, 10-30, or 15-25 nucleotides in length.

25. The guide RNA according to claim 23, wherein the transposon-associated motif comprises a nucleotide sequence as shown in the following formula:(X17)h(X18)(X19)A(X20)wherein,h is the number of nucleotides;A is an adenine deoxyribonucleotide;(X17) is any deoxyribonucleotide, and h is 0 or 1;(X18) is a cytosine deoxyribonucleotide or thymine deoxyribonucleotide;(X19) is a cytosine deoxyribonucleotide, thymine deoxyribonucleotide, or guanine deoxyribonucleotide; and(X20) is any deoxyribonucleotide.

26. A nucleic acid, wherein, the nucleic acid encodes the nuclease according to claim 12.

27. A nucleic acid construct, comprising the nucleic acid according to claim 26.

28. (canceled)29. The nucleic acid construct according to claim 27, wherein the nucleic acid construct is modified by 5′-end capping and / or 3′-end polyadenylating.

30. The nucleic acid construct according to claim 27, wherein the nucleic acid construct is modified by thiophosphate bond modification, 2′-MOE (2-O-(2-methoxyethyl)), PNA (peptide nucleic acid), GNA (glycerol nucleic acid), LNA (locked nucleic acid), GalNAc (N-acetylgalactosamine), LNP (lipid nano particle) PNP (peptide nanoparticles).

31. A composition, wherein, the composition includes:an IS200 / IS605 family nuclease or a functional fragment thereof, or comprises a nucleic acid encoding the IS200 / IS605 family nuclease or the functional fragment thereof, and the nuclease or the functional fragment thereof has endonuclease activity; anda guide RNA, or comprises a nucleic acid encoding the guide RNA, and the guide RNA can bind to a specific nuclease.

32. The composition according to claim 31, wherein the composition is selected from at least one of the following groups (1)-(198), and any one of the following groups (1)-(198) comprises: a nuclease-related sequence and a guide RNA-related sequence,(1) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 1 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 198;(2) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 2 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 199;(3) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 3 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 200;(4) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 4 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 201;(5) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 5 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 202;(6) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 6 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 203;(7) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 7 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 204;(8) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 8 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 205;(9) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 9 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 206;(10) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 10 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 207;(11) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 11 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 208;(12) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 12 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 209;(13) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 13 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 210;(14) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 14 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 211;(15) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 15 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 212;(16) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 16 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 213;(17) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 17 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 214;(18) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 18 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 215;(19) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 19 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 216;(20) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 20 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 217;(21) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 21 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 218;(22) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 22 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 219;(23) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 23 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 220;(24) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 24 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 221;(25) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 25 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 222;(26) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 26 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 223;(27) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 27 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 224;(28) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 28 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 225;(29) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 29 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 226;(30) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 30 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 227;(31) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 31 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 228;(32) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 32 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 229;(33) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 33 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 230;(34) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 34 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 231;(35) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 35 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 232;(36) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 36 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 233;(37) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 37 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 234;(38) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 38 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 235;(39) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 39 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 236;(40) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 40 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 237;(41) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 41 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 238;(42) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 42 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 239;(43) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 43 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 240;(44) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 44 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 241;(45) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 45 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 242;(46) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 46 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 243;(47) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 47 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 244;(48) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 48 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 245;(49) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 49 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 246;(50) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 50 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 247;(51) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 51 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 248;(52) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 52 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 249;(53) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 53 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 250;(54) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 54 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 251;(55) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 55 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 252;(56) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 56 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 253;(57) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 57 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 254;(58) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 58 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 255;(59) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 59 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 256;(60) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 60 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 257;(61) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 61 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 258;(62) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 62 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 259;(63) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 63 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 260;(64) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 64 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 261;(65) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 65 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 262;(66) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 66 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 263;(67) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 67 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 264;(68) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 68 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 265;(69) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 69 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 266;(70) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 70 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 267;(71) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 71 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 268;(72) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 72 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 269;(73) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 73 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 270;(74) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 74 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 271;(75) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 75 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 272;(76) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 76 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 273;(77) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 77 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 274;(78) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 78 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 275;(79) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 79 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 276;(80) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 80 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 277;(81) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 81 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 278;(82) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 82 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 279;(83) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 83 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 280;(84) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 84 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 281;(85) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 85 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 282;(86) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 86 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 283;(87) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 87 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 284;(88) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 88 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 285;(89) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 89 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 286;(90) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 90 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 287;(91) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 91 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 288;(92) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 92 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 289;(93) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 93 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 290;(94) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 94 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 291;(95) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 95 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 292;(96) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 96 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 293;(97) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 97 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 294;(98) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 98 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 295;(99) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 99 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 296;(100) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 100 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 297;(101) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 101 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 298;(102) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 102 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 299;(103) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 103 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 300;(104) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 104 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 301;(105) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 105 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 302;(106) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 106 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 303;(107) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 107 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 304;(108) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 108 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 305;(109) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 109 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 306;(110) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 110 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 307;(111) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 111 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 308;(112) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 112 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 309;(113) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 113 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 310;(114) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 114 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 311;(115) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 115 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 312;(116) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 116 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 313;(117) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 117 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 314;(118) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 118 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 315;(119) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 119 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 316;(120) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 120 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 317;(121) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 121 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 318;(122) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 122 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 319;(123) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 123 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 320;(124) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 124 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 321;(125) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 125 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 322;(126) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 126 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 323;(127) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 127 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 324;(128) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 128 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 325;(129) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 129 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 326;(130) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 130 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 327;(131) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 131 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 328;(132) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 132 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 329;(133) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 133 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 330;(134) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 134 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 331;(135) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 135 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 332;(136) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 136 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 333;(137) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 137 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 334;(138) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 138 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 335;(139) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 139 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 336;(140) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 140 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 337;(141) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 141 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 338;(142) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 142 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 339;(143) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 143 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 340;(144) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 144 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 341;(145) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 145 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 342;(146) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 146 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 343;(147) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 147 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 344;(148) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 148 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 345;(149) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 149 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 346;(150) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 150 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 347;(151) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 151 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 348;(152) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 152 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 349;(153) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 153 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 350;(154) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 154 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 351;(155) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 155 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 352;(156) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 156 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 353;(157) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 157 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 354;(158) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 158 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 355;(159) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 159 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 356;(160) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 160 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 357;(161) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 161 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 358;(162) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 162 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 359;(163) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 163 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 360;(164) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 164 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 361;(165) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 165 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 362;(166) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 166 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 363;(167) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 167 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 364;(168) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 168 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 365;(169) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 169 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 366;(170) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 170 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 367;(171) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 171 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 368;(172) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 172 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 369;(173) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 173 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 370;(174) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 174 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 371;(175) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 175 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 372;(176) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 176 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 373;(177) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 177 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 374;(178) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 178 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 375;(179) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 179 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 376;(180) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 180 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 377;(181) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 181 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 378;(182) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 182 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 379;(183) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 183 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 380;(184) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 184 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 381;(185) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 185 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 382;(186) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 186 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 383;(187) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 187 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 384;(188) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 188 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 385;(189) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 189 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 386;(190) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 190 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 387;(191) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 191 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 388;(192) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 192 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 389;(193) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 193 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 390;(194) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 194 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 391;(195) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 195 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 392;(196) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 196 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 393;(197) the nuclease-related sequence is an amino acid sequence comprising the sequence as shown in SEQ ID NO: 197 or a nucleic acid encoding the amino acid sequence; and the guide RNA-related sequence is a nucleotide sequence comprising the sequence as shown in SEQ ID NO: 394;(198) a variant of any one of the aforementioned groups (1)-(197),wherein the nuclease-related sequence is the amino acid sequence of the variant of the nuclease in each group or a nucleic acid sequence encoding the variant, and the variant has a variant sequence of the aforementioned nuclease having a nuclease activity selected from the following (i)-(iii):(i) at least one of sequences obtained by performing deletion, substitution, insertion, or mutation of 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 amino acids on the amino acid sequence of the nuclease in each group;(ii) at least one of amino acid sequences having at least 70%, 80%, 90%, 95% or 99% identity to the amino acid sequence as shown in any one of SEQ ID NOs: 1-197; and(iii) at least one of sequences obtained by further fusing the amino acid sequence as shown in any one of SEQ ID NO: 1-197 with other sequences.

33. The composition according to claim 32, wherein the guide RNA-related sequence further comprises a targeted sequence that can recognize a targeting gene adjacent to a transposon-associated motif.

34. The composition according to claim 33, wherein the targeted sequence is of at least one of 10-50, 10-40, 10-30, or 15-25 nucleotides in length.

35. The composition according to claim 33, wherein the transposon-associated motif comprises a nucleotide sequence as shown in the following formula:(X17)h(X18)(X19)A(X20)wherein,h is the number of nucleotides;A is an adenine deoxyribonucleotide;(X17) is any deoxyribonucleotide, and h is 0 or 1;(X18) is a cytosine deoxyribonucleotide or thymine deoxyribonucleotide;(X19) is a cytosine deoxyribonucleotide, thymine deoxyribonucleotide, or guanine deoxyribonucleotide; and(X20) is any deoxyribonucleotide.

36. A recombinant vector, wherein, the recombinant vector comprises a nucleic acid encoding the nuclease according to claim 12.

37. The recombinant vector according to claim 36, wherein the recombinant vector includes a recombinant cloning vector, a recombinant eukaryotic expression plasmid, or a recombinant viral vector.38.-39. (canceled)40. A recombinant host cell, wherein, the recombinant host cell comprises the nuclease according to claim 12.

41. The recombinant host cell according to claim 40, wherein the recombinant host cell includes an animal cell, a plant cell, an algal cell, a fungal cell, a yeast cell, or a bacterial cell.42.-44. (canceled)45. A method for deleting, replacing or inserting a targeting gene of a host cell, wherein the method comprises: delivering the nuclease according to claim 12, or a nucleic acid encoding the nuclease according to claim 12 into a host cell.

46. (canceled)47. The method according to claim 45, wherein the delivery method includes cationic liposome delivery, lipoid nanoparticulate delivery, cationic polymer delivery, vesicle-exosome delivery, gold nanoparticulate delivery, polypeptide and protein delivery, retrovirus delivery, lentivirus delivery, adenovirus delivery, adeno-associated virus delivery, electroporation, agrobacterium infection, or gene gun.48.-56. (canceled)57. A kit, wherein, the kit comprises the nuclease according to claim 12.

58. A nucleic acid, wherein, the nucleic acid encodes the guide RNA according to claim 19.

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